Polypeptides, chimeric nuclease, formulations comprising same, and methods for editing genomic DNA

Abstract

The invention relates to a polypeptide, a chimeric nuclease, a preparation containing the same and a method for editing genomic DNA. The present invention provides a polypeptide comprising the complete amino acid sequence of SEQ ID NO: 14 or a fragment thereof, provided that the fragment contains a Glu10 mutation. The invention also provides a chimeric nuclease. The chimeric nuclease comprises a modified I-Tevl nuclease structural domain, a linker, RNA (Ribonucleic Acid) guided nuclease staphylococcus aureus Cas9 and guide RNA. The invention also provides a method for editing genomic DNA and a method for deleting DNA molecules with limited length. The method provided by the invention edits a gene by administering a chimeric nuclease to a cell or organism without the use of a viral vector.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of May 4, 2020, an application number of 2020800458884, and an invention title of "Lipid-Encapsulated Dual-Cleaving Endonucleases for DNA and Gene Editing". Technical Field

[0002] The present invention belongs to the field of gene editing, and relates to a polypeptide. The present invention also relates to a chimeric nuclease and a pharmaceutically acceptable preparation comprising the polypeptide, as well as methods for editing genomic DNA and methods for deleting DNA molecules of a defined length. Background Art

[0003] It is estimated that there are 5,000 - 10,000 monogenic diseases, which are defined as genetic disorders caused by a single gene mutation. These diseases typically occur in childhood and result in a variety of conditions, and sometimes even premature death. It is estimated that in total approximately 6% of the population will be affected by these diseases at some stage in their lives. The diagnosis and treatment of these diseases remain largely inadequate, and care is mainly palliative, focusing on disease management but not addressing the underlying genetic defect. There are also many more diseases in which gene mutations contribute to the pathogenesis of these diseases.

[0004] Gene editing is a gene therapy method that relies on engineering nucleases to recognize and cleave specific DNA sequences, and then utilizes the innate cellular DNA repair pathways, namely non-homologous end joining (NHEJ) and homology-directed repair (HDR), to introduce targeted modifications in the genome. Four nuclease families have been used in this context: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-associated RNA-guided Cas9 (CRISPR-Cas9) nucleases. These can be engineered to precisely introduce double-strand breaks at the target locus of interest. Gene editing opens up the possibility of permanently modifying genomic sequences of interest by achieving targeted disruption, insertion, excision, and correction both ex vivo and in vivo settings. While these advances hold promise for revolutionizing the field, current gene editing methods are limited by the modification efficiency, safety issues related to nuclease specificity, and the delivery of gene editing tools to the target cell type.

[0005] As a component of the type II CRISPR system that constitutes the bacterial innate immune system, the Cas9 (CRISPR-associated) protein has caused a paradigm shift in the field of genome editing due to its ease of use. Programming Cas9 to cut a desired sequence is a simple matter of altering the sequence of the Cas9-associated guide RNA to be complementary to the target site. The simplicity of Cas9 targeting programming contrasts with the more intensive protein engineering required for other reagents (zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs)). Cas9, as well as proteins from the type III CRISPR system, have been used in numerous genome editing applications in a variety of organisms and are now entering the realm of human therapeutic applications.

[0006] Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene that encodes an epithelial anion channel. The CFTR protein, cystic fibrosis transmembrane conductance regulator, is found in a wide range of organs including the pancreas, kidney, liver, lung, gastrointestinal tract, and reproductive tract, making CF a multi-organ disease. Mutations in CFTR result in poor ion transport and fluid retention, leading to the prominent clinical manifestations of abnormally thickened lung mucus and pancreatic insufficiency. In the lung, dysfunctional CFTR impedes mucociliary clearance, rendering the organ vulnerable to bacterial infection and inflammation, ultimately leading to airway obstruction, respiratory failure, and premature death. CF remains the most common and lethal genetic disease in the Caucasian population, with an estimated 70,000 - 100,000 affected individuals worldwide, highlighting the real need for better treatment methods.

[0007] A major challenge in developing CF treatment strategies is the wide diversity of mutation types. The ΔF508 (deletion of phenylalanine at codon 508) mutation has a prevalence >80% in CF patients and is by far the most common, but over 1,990 harmful CFTR mutations have been described. These mutations result in premature stop codons, abnormal splicing, incorrect protein folding or trafficking to the cell surface, and a dysfunctional CFTR with limited channel-opening ability. Drug interventions have targeted several of these processes, and while drug administration has therapeutic effects in some gating mutation types, the common ΔF508 still requires more effective treatment. However, drug progress in treating CF has not addressed mutations caused by abnormal splicing or premature stop codons; in these cases, gene editing may be most beneficial.

[0008] Similarly, in Western populations, approximately 15% of non-small cell lung cancer (NSCLC) patients carry activating mutations in the epidermal growth factor receptor (EGFR) gene in their tumors.

[0009] Existing gene editing technologies, such as CRISPR-Cas9 (and fusions with Cas9), meganucleases, zinc finger proteins, type IIS restriction endonucleases (FokI and fusions with FokI), and TALENs, have limited ability to introduce specific-length gene deletions or accurately repair target genes in a sufficient number of cells, aspects that are meaningful for use as therapeutic agents for treating many genetic diseases. In addition, for highly programmable RNA-guided nucleases, such as monomeric Cas9, studies have shown that the specificity to predictably bind, cleave, and repair only its target site is limited, raising concerns about potentially harmful changes to the cellular genomic DNA that could inadvertently lead to secondary diseases in patients. Finally, most nucleases are delivered in viral vectors. Viral vectors have the potential for: immunity in many populations; immunogenicity after treatment; and genotoxicity. Currently, there is no non-viral delivery method that can safely deliver nucleases to target cells and allow for controlled administration of nucleases in vivo.

[0010] There remains an unmet need to improve the existing gene editing technologies to address the above problems to make gene editing technologies more efficient and effective. Summary of the Invention

[0011] The present invention relates to a chimeric nuclease comprising a modified I-TevI nuclease domain, preferably with Met 1 deleted and having Lys 26 (which is Lys 27 in the non-truncated version of I-TevI) and / or Cys 39 (which is Cys 40 in the non-truncated version of I-TevI) modifications; a linker, especially SEQ ID NO: 7-12 or a fragment thereof and / or containing one or more of the following mutations: Thr 95 (referenced to full-length I-TevI), Val 117 , Lys 135 , Gln 158 or Asn 140 ; and a modified RNA-guided nuclease Staphylococcus aureus Cas9, which may be a wild-type or modified version, preferably containing its Glu 10 or Ala 557 mutation, wherein the I-TevI polypeptide comprises the complete amino acid sequence of SEQ ID NO: 6 or a fragment thereof; and a guide RNA, especially SEQ ID NO: 15, 16 or 21 or a fragment thereof, which targets the Cas9 domain, and a pharmaceutically acceptable formulation comprising the chimeric nuclease, cationic and / or neutral lipid nanoparticles, optionally a DNA-binding compound, especially GL67 (N4 -cholesteryl-spermine), and a pharmaceutically acceptable carrier thereof.

[0012] In a further embodiment of the present invention, the lipid nanoparticles in the formulation may contain exogenous donor DNA.

[0013] Another embodiment of the present invention relates to a method of editing genes in vivo without using a viral vector by administering a chimeric nuclease to a cell or an organism using a controlled dose.

[0014] Another embodiment of the present invention relates to a method of deleting a defined length of a DNA molecule or replacing a selected sequence in a DNA molecule by in vivo delivery of a chimeric nuclease to an entire organism or ex vivo delivery to isolated cells in culture, wherein the cells are mammalian cells, bacteria, insect cells or plant cells.

[0015] In yet another embodiment, the novel chimeric nuclease targets two independent target sites on a selected DNA molecule, cuts at one target site or at both target sites, and forms a fragment with a length of 30 to 36 nucleotides.

[0016] In a further example, the novel purified chimeric nuclease further comprises a guide RNA.

[0017] Another aspect of the present invention is to form particles with a diameter of about 100 nM containing excipients using an extrusion process, wherein the excipients are selected from the group consisting of: polysorbate, polyphosphate, calcium chloride, sodium chloride, sodium citrate, sodium hydroxide, sodium phosphate, sodium ethylenediaminetetraacetate, potassium chloride, potassium phosphate and starch or a mixture of these substances, so that the novel chimeric nuclease can be administered to a patient using a nebulizer containing the formulation.

[0018] In a preferred embodiment, the present invention relates to a method of treating a lung-related disease in a patient in need thereof by administering a novel chimeric nuclease that modifies the DNA of lung epithelial cells, wherein the chimeric nuclease replaces the CFTRδF508 mutation in the CFTR gene to treat cystic fibrosis, or cuts the deletion of exon 19 of EGFR to treat non-small cell lung cancer.

[0019] In yet another embodiment, the present invention relates to a chimeric nuclease comprising a modified I-TevI nuclease domain, a linker and a modified RNA-guided nuclease Staphylococcus aureus Cas9, wherein the RNA-guided nuclease Staphylococcus aureus Cas9 contains Ala 10 、Ala 557 or Ala 580 mutations and targets the deletion of exon 19 of the EGFR gene.

[0020] In a further embodiment, the guide RNA targets a specific CFTR gene sequence to excise the CFTR ΔF508 mutation or a specific EGFR gene sequence containing an EGFR exon 19 deletion mutation.

[0021] The present invention also encompasses linkers comprising SEQ ID NO: 7-12 or fragments thereof, and modified donor DNA molecules selected from the group consisting of: a linear single-stranded DNA comprising homologous regions flanking the site targeted and / or cleaved by a chimeric nuclease; a linear double-stranded DNA comprising homologous regions flanking the site targeted and / or cleaved by a chimeric nuclease; a double-stranded DNA of the same length comprising DNA ends complementary to the DNA ends cleaved by the chimeric nuclease; a circular double-stranded DNA comprising homologous regions flanking the site targeted and / or cleaved by a chimeric nuclease; and a circular double-stranded DNA comprising an I-TevI target site and a Cas9 target site, wherein the product cleaved from the double-stranded DNA contains ends complementary to the ends cleaved by the chimeric nuclease.

[0022] In a further example consisting of a chimeric nuclease, the chimeric nuclease comprises a modified GIY-YIG nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9 or Streptococcus pyogenes Cas9 or an S. pyogenes Cas9 variant EQR, the S. pyogenes Cas9 variant EQR containing a Glu 10 mutation (SEQ ID NO: 19) and / or an Ala 840 mutation and / or a mutation that cleaves the sugar-phosphate backbone of the target DNA on one strand of the target DNA, wherein the GIY-YIG nuclease domain is selected from the gene family consisting of I-Bmol and Eco29kI.

[0023] In yet a further embodiment, the invention includes a chimeric nuclease comprising a modified I-TevI nuclease domain, a linker, and a modified nuclease or DNA targeting domain, wherein the modified nuclease or DNA targeting domain is selected from the group consisting of: LAGLIDADG, His-Cys Box, H-N-H, PD-(D / E)xK, and Vsr-like mega nucleases, zinc finger nucleases, CRISPR proteins, and DNA binding domains, the CRISPR proteins are selected from the group consisting of scCas9 (Streptococcus canis), fnCas9 (Francisella novicida), cjCas9 (Campylobacter jejuni), Cpf1 (Lachnospiraceae bacterium), Cas12a (Acidaminococcus Sp), Cas13a (Leptorichia shahii), and Cas3 (Streptococcus thermophilus), and the DNA binding domains are selected from the group consisting of zinc finger motifs and TALE activation domains.

[0024] In a still further example, the invention encompasses a modified RNA-guided nuclease, Staphylococcus aureus Cas9, and a guide RNA, wherein the guide RNA contains a sequence targeting a gene polymorphism, different sequences in the CFTR or EGFR gene, a sequence for re-targeting a nuclease, a bridging nucleic acid, and / or a mixture of guide RNAs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic illustration of a lipid-encapsulated dual-cut nuclease (TevCas9) after its preparation [components not to scale]. The I-TevI domain 10 is linked to the RNA-guided nuclease (Cas9) domain 12 by a linker domain 11. In a preferred embodiment, the formed particles also contain a guide RNA 13 and a donor DNA 14. The foregoing nuclease is contained within a lipid particle 15 that has been formed into a sphere using an extrusion process.

[0026] Figure 2Figure depicting the internalization of lipid-encapsulated TevCas9 into cells and the nucleus to reach its target DNA. As shown in A, by in vivo or ex vivo administration, cell 20 or multiple cells 20 are exposed to novel lipid-encapsulated nuclease particles 21 containing TevCas9 25. As shown in B, the lipid-encapsulated nuclease particles 21 are endocytosed into cell 20. The endosome 22 undergoes a maturation process in the cytosol and is targeted for degradation (C). In some cases, TevCas9 25 can escape from the endosome 22 and enter the cytosol (D). In eukaryotes, the nuclease (TevCas9) 25 is targeted to the nucleus 23 of cell 20 via one or more nuclear localization sequences (“NLS”). As depicted in E, via its nuclear localization sequence, TevCas9 25 can enter the nucleus 23, and when in the nucleus 23, the TevCas9 nuclease 25 binds and cleaves 26 the target genomic DNA 24 sequence.

[0027] Figure 3 Figure depicting the mechanism by which lipid-encapsulated TevCas9 modifies target DNA. As shown in A, the I-TevI domain 27 targets the I-TevI target sequence 29. The linker domain 30 connects the I-TevI domain 27 to the Cas9 domain 28 that targets the Cas9 target sequence 31. The gene mutation 32 is surrounded or in proximity to the I-TevI target sequence 29 and the Cas9 target sequence 31. As shown in B, TevCas925 cleaves the target sequence, leaving a deletion product 34 of predictable size with non-complementary DNA ends 35, 36. C illustrates that in the presence of single-stranded donor DNA with homologous arms 37, cell 20 can insert the donor DNA37 sequence near the cleavage site via the homologous directed repair (HDR) pathway 38. D illustrates that in the presence of donor DNA 39 with DNA ends compatible with the DNA ends cleaved by TevCas9 25, cell 20 can use the non-homologous end joining (NHEJ) pathway 40 to insert the donor DNA sequence 39 between the cleavage sites by directed ligation. In the absence of donor DNA, cell 20 can ligate the DNA ends via the NHEJ pathway 40 (E).

[0028] Figure 4 : A demonstrates the in vitro cleavage of CFTR DNA substrate by TevCas9 using the guide targeting the CFTR gene in SEQ ID 15. B is a cell transfected with a plasmid DNA version of TevCas9 fused to a cleavable GFP tag and imaged on a Cytation 5 (Biotek Instruments Inc, VT, USA) using phase contrast and GFP imaging 48 hours after treatment. Genomic DNA was extracted from the collected cells and the editing at the CFTR gene was detected by PCR amplification and T7 endonuclease I cleavage assay.

[0029] Figure 5 It was demonstrated that TevCas9 targeting the CFTR ΔF508 mutation using the guide in SEQ ID 21 cleaved the DNA substrate containing the CFTR ΔF508 mutation in vitro, but did not cleave the substrate containing the wild-type CFTR sequence.

[0030] Figure 6 : A shows that the saCas9 D10E mutation slows down the conversion of nicked supercoiled DNA to linear DNA. B demonstrates that on the linear EMX1 DNA substrate, the saCas9 D10E (D10E) ribonucleoprotein complex (RNP) cleaves the target substrate to a level similar to that of saCas9 wild-type (WT). The editing level of saCas9 D10E at computationally predicted off-targets is lower than that of wild-type saCas9 at the same off-targets.

[0031] Figure 7 : A is a schematic diagram of the spacing of the I-TevI site in EGFR exon 19 deletion and wild-type (WT) EGFR. B and C demonstrate that TevCas9 containing a nick mutation in Cas9 (H557A) targeting EGFR using the guide RNA in SEQ ID 16 cleaves the EGFR exon 19 deletion DNA substrate at a rate 4 times faster than that of wild-type EGFR. D is an image of HCC827 cells carrying the EGFR exon 19 deletion mutation treated with TevCas9 targeting EGFR being selectively killed compared to NuLi-1 cells carrying wild-type EGFR (WT). Detailed Description

[0032] Definitions and Acronyms

[0033] For convenience, certain terms employed in the specification, examples, and appended claims are collected herein. These definitions should be read in light of the disclosure and understood by those of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0034] The article "a / an" is used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. The term "and / or" as used herein is defined to have the possibility of one or the other or both. By way of example, "A and / or B" provides the situation of having only A, only B, or the combination of A and B. If the claim is written as A and / or B and / or C, then the composition may include only A, only include B, only include C, include A and B but not C, include B and C but not A, include A and C but not B, or include all three A, B, and C as components.

[0035] The term "bioavailable" is well-known in the art and refers to a form of the present disclosure that allows a portion of it or the amount administered to be absorbed, incorporated, or otherwise physiologically available to the subject or patient to whom it is administered.

[0036] As used herein, the term "exogenous donor DNA" refers to any sequence of DNA that is wholly or partially different from the original target DNA sequence.

[0037] As used herein, the term "flexible linker" refers to the situation when the RNA-guided nuclease domain (Cas9) binds to the target DNA sequence. The amino acid linker domain ensures the mobility of the I-TevI domain to allow recognition, binding, and cleavage of its target sequence under cellular physiological conditions (typically: pH ~ 7.2, temperature: ~ 37 °C, [K+] ~ 140 mM, [Na+] ~ 5 - 15 mM, [Cl-] ~ 4 mM, [Ca++] ~ 0.0001 mM). The length of the amino acid linker can affect the preferred number of nucleotides between the Cas9 target site and the I-TevI target site. Certain amino acids in the linker can also make specific contacts with the DNA sequence targeted by TevCas9. These linker-DNA contacts can affect the flexibility of the I-TevI domain. Substituting amino acids in the linker domain can affect the ability of the linker domain to make contacts with the DNA.

[0038] As used herein, the term "comprising" is used to mean "including but not limited to". "Comprising" and "including but not limited to" can be used interchangeably.

[0039] The terms "inhaled administration", "inhale / inhaled / inhalation", or "inhalation therapy" can be used interchangeably and, as used herein, include the administration of appropriately sized particles that are substantially evenly distributed to the respiratory epithelium of the nose, the central airways, the peripheral portions of the lung, and / or the alveolar regions of the lung or by intratracheal instillation. Such particles can be introduced to the patient and / or generated using an appropriate device, preferably a nebulizer.

[0040] The terms "patient", "subject", or "host" to be treated by the present method can mean a human or a non-human animal. Non-human animals include companion animals (such as cats, dogs) and animals raised for food (i.e., food animals), such as cows, pigs, and chickens.

[0041] The term "pharmaceutically acceptable carrier" is well known in the art and refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in transporting or conveying any composition or its components from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the composition and its components and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as dextrose, lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as microcrystalline cellulose, sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), and cellulose acetate; (4) glycols, such as propylene glycol; (5) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (6) esters, such as ethyl oleate, glyceryl behenate, and ethyl laurate; (7) buffers, such as monohydrogen phosphate and dihydrogen phosphate, Tris / boric acid / EDTA, and Tris / acetic acid / EDTA; (8) pyrogen-free water; (9) isotonic saline; (10) Ringer's solution; (11) ethanol; (12) phosphate buffer solution; (13) polysorbate; (14) polyphosphate; and (15) other non-toxic compatible substances used in pharmaceutical formulations. The disclosed excipients can be used for more than one function. For example, a solubilizer can also be a suspending aid, emulsifying agent, preservative, etc.

[0042] In certain preferred embodiments, the pharmaceutically acceptable excipient is a crystalline bulking excipient. As used herein, the term "crystalline bulking excipient" or "crystalline bulking agent" means an excipient that provides volume and structure to the lyophilized cake. These crystalline bulking agents are inert and do not react with proteins or nucleic acids. In addition, the crystalline bulking agent is capable of crystallizing under lyophilization conditions. Examples of suitable crystalline bulking agents include hydrophilic excipients such as water-soluble polymers; sugars such as mannitol, sorbitol, xylitol, glucitol, dulcitol, inositol, arabitol, allitol, galactitol, iditol, alloxan, maltitol, fructose, sorbose, glucose, xylose, trehalose, allose, dextran, altrose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, sucrose, maltose, lactose, lactulose, fucose, rhamnose, melezitose, maltotriose, raffinose, atritol, their optional active forms (D or L) and the corresponding racemates; inorganic salts, both mineral salts and organomineral salts, such as calcium salts such as lactate, gluconate, glycerophosphate, citrate, monohydrogen phosphate and dihydrogen phosphate, succinate, sulfate and tartrate, and the above salts of aluminum and magnesium; carbohydrates such as conventional monosaccharides and disaccharides and the corresponding polyhydroxy alcohols; proteins such as albumin; amino acids such as glycine; emulsifiable fats and polyvinylpyrrolidone. Preferred crystalline bulking agents are selected from the group consisting of glycine, mannitol, dextran, dextrin, lactose, sucrose, polyvinylpyrrolidone, trehalose, glucose and combinations thereof. Particularly useful bulking agents include dextran.

[0043] As used herein, the term "pharmaceutically acceptable salt" is well known in the art and refers to relatively non-toxic inorganic acid addition salts and organic acid addition salts, or inorganic base addition salts or organic base addition salts of a compound, including for example the salts contained in the compositions of the present invention. Some examples of pharmaceutically acceptable salts include: (1) calcium chloride; (2) sodium chloride; (3) sodium citrate; (4) sodium hydroxide; (5) sodium phosphate; (6) sodium ethylenediaminetetraacetate; (7) potassium chloride; (8) potassium phosphate; and (9) other non-toxic compatible substances used in pharmaceutical formulations.

[0044] As used herein, the term "substitution" means replacing an amino acid in a sequence with a different amino acid. As used herein, the shorthand X10Y indicates that amino acid Y has "substituted" the amino acid X present at position 10 in the sequence. For example, W26C indicates that amino acid tryptophan-26 (Trp, W) has been changed to cysteine (Cys). Similarly, the symbol AA X denotes that AA is an amino acid that has replaced the amino acid present at position X. For example, Lys 26Indicates the replacement of the amino acid at position 26 in the sequence with lysine. The use of the two abbreviations is interchangeable. In addition, the use of one-letter or three-letter abbreviations for amino acids is also interchangeable.

[0045] As used herein, the term "therapeutic agent" is well known in the art and refers to any chemical or biochemical moiety that acts locally or systemically in a subject as a biologically, physiologically, or pharmacologically active substance. Examples of therapeutic agents, also known as "drugs", are described in well-known references such as the Merck Index, the Physician's Desk Reference, and The Pharmacological Basis of Therapeutics, and include, but are not limited to, pharmaceuticals; vitamins; mineral supplements; substances for treating, preventing, diagnosing, curing, or alleviating a disease or disorder; substances that affect the structure or function of the body; or prodrugs, which become biologically active or more active when placed in a physiological environment.

[0046] As used herein, the term "therapeutic effect" is well known in the art and refers to a local or systemic effect in an animal, particularly a mammal, and more particularly a human, caused by a pharmacologically active substance. Thus, the term means any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease or for enhancing a desired physical or mental advancement and / or condition in an animal or human. The phrase "therapeutically effective amount" means an amount of such substance that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The therapeutically effective amount of such substance will vary depending on the subject being treated and the disease condition, the weight and age of the subject, the severity of the disease condition, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art. For example, certain compositions of the present invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0047] As used herein, the term "treatment" includes any effect that results in the amelioration of a disorder, disease, affliction, etc., such as alleviation, reduction, modulation, or elimination. As used herein, "treatment" can include both prophylactic treatment and therapeutic treatment. For example, therapeutic treatment can include delaying, inhibiting, or arresting the progression of cystic fibrosis or non-small cell lung cancer, reducing or eliminating symptoms associated with cystic fibrosis or non-small cell lung cancer. Prophylactic treatment can include preventing, inhibiting, or delaying the onset of cystic fibrosis or non-small cell lung cancer.

[0048] As used herein, "effective amount" means an amount sufficient to elicit a desired response. In the present invention, the desired biological response is the treatment of cystic fibrosis and / or non-small cell lung cancer (NSCLC).

[0049] As used herein, "buffer" is any combination of acids or salts that is pharmaceutically acceptable and capable of maintaining the composition of the present invention within a desired pH range. The buffer in the disclosed composition maintains the pH at about 2 to about 8.5, about 5.0 to about 8.0, about 6.0 to about 7.5, about 6.5 to about 7.5, or about 6.5. Suitable buffers include any pharmaceutically acceptable buffer capable of maintaining the above pH ranges, such as, for example, acetate, tartrate, phosphate, or citrate buffers. In one embodiment, the buffer is a phosphate buffer. In another embodiment, the buffer is an acetate buffer. In one embodiment, the buffer is disodium hydrogen phosphate, sodium chloride, potassium chloride, and potassium dihydrogen phosphate.

[0050] In the disclosed composition, the concentration of the buffer is generally from about 0.1 mM to about 1000 mM, about 0.2 mM to about 200 mM, about 0.5 mM to about 50 mM, about 1 mM to about 10 mM, or about 6.0 mM.

[0051] As used herein, "antimicrobial agent" is a pharmaceutically acceptable preservative suitable for administration to a subject, which inhibits, prevents, or delays the growth of microorganisms in the composition of the present invention, including, for example, bacteria, viruses, and fungi. Suitable antimicrobial agents for the compositions and methods of the present invention include, but are not limited to, cresol, benzyl alcohol, phenol, benzalkonium chloride, benzethonium chloride, chlorobutanol, phenethyl alcohol, methylparaben, propylparaben, thimerosal, phenylmercuric nitrate, and phenylmercuric acetate. In one embodiment, the antimicrobial agent is m-cresol, chlorocresol, or phenol. In another embodiment, the antimicrobial agent is chlorocresol or phenol. In another embodiment, the antimicrobial agent is phenol. As used herein, an effective amount of the antimicrobial agent is an amount effective to inhibit, prevent, or delay the growth of microorganisms in the composition of the present invention, including, for example, bacteria, viruses, and fungi. In the composition of the present invention, the amount of the antimicrobial agent is generally from about 0.1 to about 20 mg / ml, about 0.2 to about 30 mg / ml, about 0.2 to about 10 mg / ml, about 0.25 to about 5 mg / ml, about 0.5 to about 50 mg / ml, about 1 to about 10 mg / ml, about 3 mg / ml, or about 5 mg / ml.

[0052] The compositions of the present invention can also be lyophilized using lyophilization techniques known in the art and stored in powder form, which can be reconstituted prior to administration. As used herein, the term "lyophilization" is a freeze-drying or dehydration technique that involves removing solvents, preferably water-miscible solvents, more preferably water, from the compositions of the present invention, typically by sublimation under high vacuum while the composition is in a frozen state. Generally, lyophilization is carried out in a lyophilization device (lyophilizer), which includes a drying chamber with variable temperature control, a condenser for collecting water, and a vacuum system for reducing the pressure in the drying chamber.

[0053] As used herein, the term "lyophilized composition" means the solid residue or powder produced or remaining after the lyophilization procedure as defined above. The lyophilized compositions of the present invention generally also contain pharmaceutically acceptable excipients. As used herein, the term "pharmaceutically acceptable excipient" refers to a substance added to the solution prior to lyophilization to enhance characteristics such as color, texture, strength, and volume of the lyophilized cake. Pharmaceutically acceptable excipients can be, for example, buffers and pH regulators, crystalline swelling excipients, stabilizers, and tonicity enhancers.

[0054] As used herein, a stabilizer is a composition that maintains the chemical, biological, or stability of the chimeric nuclease. Examples of stabilizers include polyols, which include saccharides, preferably monosaccharides or disaccharides, such as glucose, trehalose, raffinose, or sucrose; sugar alcohols, such as mannitol, sorbitol, or inositol, polyhydric alcohols, such as glycerol or propylene glycol, or mixtures thereof, and albumin.

[0055] Pharmaceutically acceptable salts are salts suitable for administration to a subject such as a human. The chimeric nucleases of the present invention may have one or more protons that are sufficiently acidic and which may react with a suitable organic or inorganic base to form a base addition salt. Base addition salts include base addition salts derived from: inorganic bases such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.; and organic bases such as alkoxides, alkylamides, alkylamines, and arylamines, etc. Thus, such bases useful for preparing the salts of the present invention include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, etc. The chimeric nucleases of the present invention having a group with sufficient basicity such as an amine may react with an organic or inorganic acid to form an acid addition salt. Acids commonly used to form acid addition salts from compounds having basic groups are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of such salts include sulfates, bisulfates, hydrogen sulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, caprates, octylates, acrylates, formates, isobutyrates, hexanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, etc.

[0056] Although specific embodiments of the invention have been discussed, the above description is illustrative and not restrictive. After reading this specification, many variations of the invention will be apparent to those skilled in the art. The full scope of the invention should be determined by reference to the claims and their full scope of equivalents and the specification and these variations.

[0057] Unless otherwise indicated, all numbers expressing component amounts, reaction conditions, etc. used in the specification and claims should be understood to be modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.

[0058] The foregoing discussion is intended to illustrate the principles of the present invention and its various embodiments. Once the above disclosure is fully understood, many variations and modifications will become apparent to those skilled in the art. The appended claims are intended to be construed to cover all such variations and modifications.

[0059] Abbreviations

[0060] The abbreviations used herein are defined as follows:

[0061] AA Amino acid

[0062] Cas9 CRISPR-associated protein 9

[0063] CF Cystic fibrosis

[0064] CFTR Cystic fibrosis transmembrane conductance regulator

[0065] cjCas9 Campylobacter jejuni Cas9

[0066] Cpf1 CRISPR 1 from Prevotella and Francisella

[0067] CRISPR Clustered regularly interspaced short palindromic repeats

[0068] DLS Dynamic light scattering

[0069] DMEM Dulbecco's Modified Eagle Medium

[0070] DMPE 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine

[0071] DNA Deoxyribonucleic acid

[0072] DOAB Dioctadecyldimethylammonium bromide

[0073] DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine

[0074] DPPC Dipalmitoylphosphatidylcholine

[0075] E.Coli Escherichia coli

[0076] EDTA Ethylenediaminetetraacetic acid

[0077] EGFR Epidermal growth factor receptor

[0078] ELISA Enzyme-linked immunosorbent assay

[0079] fnCas9 Francisella novicida Cas9

[0080] HDR Homology Directed Repair

[0081] IMAC Immobilized Metal Affinity Chromatography

[0082] IPTG Isopropyl β-D-1-thiogalactopyranoside

[0083] MPEG-5000-DMPE N-(Carboxy-Methoxypolyethylene Glycol 5000)-1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine

[0084] NSCLC Non-Small Cell Lung Cancer

[0085] NHEJ Non-Homologous End Joining

[0086] NLS Nuclear Localization Signal

[0087] PC Phosphatidylcholine

[0088] PCR Polymerase Chain Reaction

[0089] PE Phosphoethanolamine

[0090] RNA Ribonucleic Acid

[0091] saCas9 Staphylococcus aureus Cas9

[0092] scCas9 Streptococcus canis Cas9

[0093] SDS Sodium Dodecyl Sulfate

[0094] spCas9 Streptococcus pyogenes Cas9

[0095] TALEN Transcription Activator-Like Effector Nuclease

[0096] TEV Tobacco Etch Virus

[0097] TevCas9 Engineered I-TevI Domain, Linker Peptide, and Engineered RNA-Guided Nuclease Staphylococcus aureus Cas9

[0098] ZFN Zinc Finger Nuclease

[0099] The inventors have discovered a chimeric nuclease that comprises a modified version of the I-TevI domain, a linker peptide, and a modified version of the RNA-guided nuclease Staphylococcus aureus Cas9 (“saCas9”) (hereinafter referred to as “TevCas9”), which, when mixed with lipid nanoparticles with or without exogenous donor DNA, when delivered to cells, replaces a DNA sequence in the presence of exogenous donor DNA or causes a defined-length DNA deletion in the absence of exogenous donor DNA. This novel chimeric nuclease has been shown to be capable of editing genes in human cells as well as in cells of other organisms such as bacteria, yeast, insects, plants, or other mammals, either in whole organisms (in vivo) or in isolated cell cultures (ex vitro).

[0100] The novel chimeric nuclease discovered by the present inventors has the following advantages in particular compared to existing gene editing techniques and methods.

[0101] a. The nuclease is a modified version of the TevCas9 nuclease that is capable of targeting two independent target sites as a single protein and cleaving DNA at one or both of these sites. By modifying one or more of the I-TevI domain, the linker domain, the Cas9 domain, or the guide RNA that directs the Cas9 domain to its target sequence, it can be reprogrammed to many different target DNA sequences;

[0102] b. If the nuclease cleaves at two sites, then it cleaves off a precisely defined length of DNA (∼30-36 bases, depending on the sites targeted by I-TevI and Cas9);

[0103] c. The Cas9 domain contains a mutation (D10E) that has been rationally designed to modify Cas9 nuclease activity and / or increase the specificity of the Cas9 domain for its target binding site;

[0104] d. In the presence of exogenous donor DNA, the present invention is designed to replace a higher percentage of the target DNA sequences in cells compared to the prior art or practice;

[0105] e. The nuclease can be purified as a single continuous protein bound to the guide RNA, thereby simplifying manufacture;

[0106] f. Lipid nanoparticles allow for non-viral delivery to target cells with high efficiency and low toxicity, thereby allowing for controlled administration of the nuclease. Although there are other lipid-based nuclease delivery techniques, none are compositions suitable for in vivo use;

[0107] g. Lipid nanoparticles are also designed for delivery of the nuclease by nebulization (inhalation);

[0108] h. A version of the nuclease targets and cleaves the CFTR gene to correct the CFTRΔF508 mutation for the treatment of cystic fibrosis (SEQ ID NO 1); and

[0109] i. Another version of the nuclease is designed to target and cleave clinically relevant EGFR exon 19 deletion mutations (SEQ ID NOs 2-4), which are present in a variety of cancers, including non-small cell lung cancer (NSCLC).

[0110] GIY-YIG nucleases such as I-TevI fused to a DNA-binding domain via a flexible linker are known (WO2014 / 121222). A previous version of the dual-cutting TevCas9 has been described, which contains amino acids 1-92 of the wild-type I-TevI nuclease domain, a linker region containing amino acids 93-169 of the I-TevI linker region, and Streptococcus pyogenes Cas9 (“spCas9”) (Wolfs JM et al., (2016), ‘Biasing Genome-Editing Events Toward Precise Length Deletions with an RNA-Guided TevCas9 Dual Nuclease,’ Proc Natl Acad Sci USA, 113(52):14988-93). The chimeric nucleases of the present invention comprise the following:

[0111] i. The I-TevI nuclease domain, which binds to a new target sequence, thereby allowing targeting of clinically relevant gene sequences, such as the CFTR gene;

[0112] ii. Various flexible linker regions, which are designed to confer different DNA-binding or nuclease activities to TevCas9;

[0113] iii. The saCas9 nuclease domain (US-1988 / 065406B2). Compared to spCas9, using saCas9 produces a smaller DNA coding sequence (Tev-saCas9 is ~3.7 kilobases compared to Tev-spCas9 which is ~4.6 kilobases) and a lower molecular weight TevCas9 protein (Tev-saCas9 is ~144 kilodaltons compared to Tev-spCas9 which is ~179 kilodaltons), which is more suitable for a variety of delivery techniques; as discovered by the inventors of the claimed technology, cleavage by the saCas9 domain between the 3rd and 4th nucleotides is predictable compared to spCas9 and is more suitable for defined length deletions.

[0114] iv. A version in which the guide RNA targets a specific CFTR gene sequence near the CFTR ΔF508 mutation; and

[0115] v. A second version in which the guide RNA targets a specific EGFR gene sequence and is designed to cleave only DNA with appropriately spaced I-TevI sites and Cas9 target sites. Such appropriately spaced sites occur in certain EGFR exon 19 deletion mutations (SEQ ID NOs 2 - 4) but not in wild-type EGFR (SEQ ID NO 5);

[0116] a. The present invention includes lipid nanoparticles of certain compositions that are selectively sized to an average diameter of approximately 100 nM. These lipid nanoparticles are capable of delivering nucleases to cells with high efficiency and low toxicity;

[0117] b. Pharmaceutical formulations of lipids, nucleases, and exogenous donor DNA;

[0118] c. Pharmaceutical formulations of lipids, nucleases, and exogenous donor DNA suitable for nebulization (inhalation); and

[0119] d. A version of the present invention contains exogenous donor DNA that, when delivered together with the TevCas9 nuclease in lipid nanoparticles, is capable of integrating into the region between or around the two sites targeted by the nuclease.

[0120] The novel chimeric nuclease compositions of the present application contain different combinations of the I-TevI domain, linker domain, Cas9 domain, and guide RNA.

[0121] The version targeting the CFTR gene contains:

[0122] i. The I-TevI domain according to the amino acid sequence of SEQ ID NO:6;

[0123] ii. The linker domain according to any one of SEQ ID NOs:7 - 12;

[0124] iii. The saCas9 domain according to the amino acid sequence of SEQ ID NO:13; and iv. The guide RNA according to the RNA sequence of SEQ ID NO:15 or 21.

[0125] The version targeting the EGFR gene contains:

[0126] i. The I-TevI domain according to the amino acid sequence of SEQ ID NO:6;

[0127] ii. a linker domain having any one of the amino acid sequences according to SEQ ID NOs: 7-12;

[0128] iii. a saCas9 domain according to the amino acid sequence of SEQ ID NO: 13; and iv. a guide RNA of the RNA sequence in SEQ ID NO: 16.

[0129] The I-TevI domain of the preferred embodiment is the 93-amino acid I-TevI domain of Enterobacteriophage T4 according to the following sequence:

[0130] MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSF NKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIA (SEQ ID NO: 6)

[0131] The preferred embodiment of saCas9 is a polypeptide of 1,053 amino acids comprising the following sequence:

[0132] MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR

[0133] GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSE

[0134] EEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAEL

[0135] QLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDL

[0136] LETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNAD

[0137] LYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVN

[0138] EEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS

[0139] SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDN

[0140] QIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKK

[0141] YGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKY

[0142] LIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNK

[0143] VLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKE

[0144] YLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKS

[0145] INGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKLDKAKK

[0146] VMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDK

[0147] KPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLL

[0148] MYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIK

[0149] KIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTV

[0150] KNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYR

[0151] VIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG(SEQ ID NO:13)

[0152] The Glu-containing preferred embodiment 10 The saCas9 with the mutation is a polypeptide of 1,053 amino acids containing the following sequence (mutation underlined): MKRNYILGL EIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG (SEQ ID NO:14)

[0153] The guide RNA of the version targeting the CFTR gene comprises 101 ribonucleotides according to the following sequence:

[0154] GCGUCAUCAAAGCAUGCCAACGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU(SEQ ID NO:15)

[0155] AUAUCAUUGGUGUUUCCUAUGGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU(SEQ ID NO:21)

[0156] The guide RNA of the version targeting the EGFR gene is 101 ribonucleotides in length according to the following sequence:

[0157] AAUUUUAACUUUCUCACCUUCGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU(SEQ ID NO:16).

[0158] The linker for any of the above constructs is optionally selected from the group consisting of:

[0159]

[0160] Synthetic

[0161] Example 1: Method for manufacturing TevCas9 nuclease

[0162] The DNA coding sequences of the I-TevI domain, linker domain, and Cas9 domain mentioned above were synthesized into a continuous DNA sequence using techniques known in the art. Gene synthesis was performed by Bio Basic Inc. (Markham, Ontario, Canada). Briefly, short oligonucleotides (~50 - 60 base pairs) were synthesized that contained overlapping regions to cover the entire sequences of the I-TevI domain, linker domain, and Cas9 domain. These oligonucleotides were mixed together in blocks of the sequence to be synthesized of approximately 1 kilobase, and these ~1 kilobase blocks were synthesized using polymerase chain reaction (PCR). Then the ~1 kilobase blocks were mixed and PCR was performed to synthesize the I-TevI domain, linker domain, and Cas9 domain. Additionally, to enhance the expression of TevCas9 in E. coli and to simplify restriction enzyme digestion, the DNA sequence of TevCas9 was optimized prior to synthesis. First, the three-base pair DNA codons that are not frequently used in Escherichia coli (“E. coli”) were replaced with more frequently occurring codons (e.g., among the 6 codons encoding the amino acid arginine, the relative abundance of the codon AGG is 0.03, while the relative abundance of the codon CGT is 0.42). In total, 37% of the codons were changed to E. coli-preferred codons. Second, the content of nucleotides cytosine and guanine was increased from 39.6% to 48.6%. Third, two E. coli ribosome binding sites were removed from the sequence. Fourth, the NdeI restriction endonuclease site was removed from the internal sequence. The continuous DNA was digested with the restriction endonucleases NdeI and BamHI (New England Biolabs, Ipswich, Massachusetts, USA), whose target sites occur only once in the DNA sequence, and then inserted into a similarly digested pET-11a expression vector (EMD Millipore, Burlington, Massachusetts, USA) suitable for expressing TevCas9 in E. coli using DNA ligase (New England Biolabs, Ipswich, Massachusetts, USA). The pET-11a vector containing TevCas9 was transformed into the E. coli expression strain T7 Express (New England Biolabs #C2566, Ipswich, Massachusetts, USA), which has been optimized for the expression of proteins including nucleases. Alternatively, the E. coli expression strain BL-21(DE3) (New England Biolabs #C2527, Ipswich, Massachusetts, USA) was used. Successful transformation was confirmed by the resistance of E. coli to ampicillin or tetracycline, and the coding sequence of TevCas9 was verified by DNA sequencing of the expression vector derived from the transformed E. coli.As measured by spectrophotometry at a wavelength of 600 nM, the transformed Escherichia coli was grown to an optical density of 0.4 to 0.6 at 37 °C, and the expression of the TevCas9 protein from the pET-11a vector in the transformed E. coli expression strain was induced with IPTG at 16 °C for 10 - 12 hours. The successful expression of TevCas9 was verified by the presence of a band of approximately 150 kDa on a Coomassie-stained SDS-polyacrylamide gel in the sample of the induced material compared to the uninduced sample. The E. coli cells were harvested by centrifugation and resuspended in a lysis buffer containing 10 mM imidazole (Sigma, St. Louis, MO, USA), 300 - 500 mM sodium chloride (Sigma-Aldrich, St. Louis, MO, USA), and 50 mM sodium phosphate (dibasic) (Sigma, St. Louis, MO, USA), pH 8.0 [Buffer 1]. Alternatively, the sodium phosphate (dibasic) in Buffer 1 was replaced with 10 mM Tris hydrochloride (Sigma, St. Louis, MO, USA) at pH 8. The E. coli was lysed by homogenization, operating a high-pressure liquid homogenizer (Avestin Inc., Ottawa, Ontario, Canada) at 600 - 1000 bar, or using any other suitable lysis method known in the art, such as sonication using a sonicator (Branson Ultrasonics Corp., Danbury, CT, USA), lysozyme treatment, homogenization using a French pressure cell (Glen Mills Inc., Clifton, NJ, USA), or homogenization using a Dounce homogenizer (Corning Inc., Corning, NY, USA). The lysed material was centrifuged at 12,000 rpm for 20 - 30 minutes at 4 °C, and the supernatant containing soluble TevCas9 was used for subsequent purification steps. The pellet contained cell debris, insoluble intracellular material, and any insoluble TevCas9. The successful lysis and solubilization were verified by the presence of a band of approximately 150 kDa on a Coomassie-stained SDS-polyacrylamide gel in the supernatant sample compared to the resuspended sample of the pellet.

[0163] The TevCas9 nuclease was purified as follows:

[0164] 1. The lysate containing the nuclease was applied to an immobilized metal affinity chromatography (IMAC) column that binds the nuclease (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

[0165] 2. The IMAC column was washed with Buffer 1.

[0166] 3. The TevCas9 that remains bound to the column is eluted with a solution containing: 250 mM imidazole (Sigma, St. Louis, MO, USA), 300 mM - 500 mM sodium chloride (Sigma - Aldrich, St. Louis, MO, USA), and 50 mM disodium phosphate (Sigma, St. Louis, MO, USA), pH 7.6 - 8.0 [Buffer 2]. Alternatively, the disodium phosphate in Buffer 2 is replaced with 10 mM Tris - hydrochloride (Sigma, St. Louis, MO, USA) at pH 7.6 - 8.

[0167] 4. The eluate is treated with tobacco etch virus (TEV) protease (New England Biolabs, Ipswich, MA, USA) and incubated with an appropriate guide RNA. The guide RNA is synthesized by Integrated DNA Technology (Coralville, IA, USA).

[0168] 5. The treated eluate is re - applied to the IMAC column, and the effluent containing the TevCas9 nuclease and the guide RNA is collected.

[0169] 6. The successful purification of the TevCas9 nuclease is confirmed by the presence of a 150 - kilodalton protein band on a Coomassie - stained SDS - polyacrylamide gel. The successful co - purification of TevCas9 with the guide RNA is confirmed by: treating an eluate sample with proteinase K (New England Biolabs, Ipswich, MA, USA), then splitting the sample into two and further treating one subsample with RNase A (New England Biolabs, Ipswich, MA, USA), while the other is in a control buffer without RNase A. An RNA band of ∼100 nucleotides can be seen on a urea - polyacrylamide gel in the control sample, but not in the RNase A - treated sample.

[0170] 7. The solution containing the TevCas9 nuclease and the guide RNA is dialyzed into a solution containing phosphate - buffered saline at pH 7.4.

[0171] Example 2: Method for manufacturing lipid nanoparticles

[0172] The lipid nanoparticles of the preferred embodiment comprise one of the following mixtures:

[0173] I. Lipid nanoparticles No. 1 contain DOPE (Avanti Polar Lipids, Alabaster, AL, United States) and MPEG-5000-DMPE (Avanti Polar Lipids, Alabaster, AL, United States) at a molar ratio of 2:0.05, respectively;

[0174] II. Lipid nanoparticles No. 2 contain DPPC (Avanti Polar Lipids, Alabaster, AL, United States), cholesterol (SUPELCO, Bellefonte, PA, United States), and DOBA (Sigma, St. Louis, MO, United States) at a molar ratio of 7:2:1, respectively; and

[0175] III. Lipid nanoparticles No. 3 contain DPPC, cholesterol, and MPEG-5000-DMPE (Avanti Polar Lipids, Alabaster, AL, United States) at a molar ratio of 4:1:0.125, respectively.

[0176] The lipid nanoparticles are manufactured to have an average diameter of approximately 100 nM.

[0177] One of the lipid mixtures No. 1-3 is selected. For example, DOPE and MPEG-5000-DMPE are mixed together in an organic solvent such as chloroform at an appropriate molar ratio. Then the organic solvent is evaporated, and the dried lipid mixture is resuspended in a solution containing phosphate buffered saline at pH 7.4 using vigorous vortexing. Then the resuspended lipid mixture is extruded through a 100 nM polycarbonate membrane (T&T Scientific Corporation, Knoxville, TN, United States) equilibrated in phosphate buffered saline to produce lipid nanoparticles with an average diameter of approximately 100 nM. The solution is filter sterilized through a 0.2 μM sterile filter (VWR Scientific, Radnor, PA, United States). The average diameter and size distribution of the lipid nanoparticles are determined by dynamic light scattering (DLS) using a Zetasizer (Malvern Panalytical Limited, Malvern, UK) or other suitable techniques known in the art.

[0178] Example 3: Composition of donor DNA

[0179] The donor DNA contains a DNA sequence designed to repair a gene defect. It also contains DNA sequences not found in the target genomic DNA; these sequences do not interfere with normal gene function but are designed to knock out the I-TevI and / or Cas9 sites and / or introduce one or more DNA sequences for tracking the successful repair of the target gene. Examples of donor DNA include, but are not limited to, the following:

[0180] I. Linear single-stranded DNA of different lengths, which contains homologous regions flanking the site targeted / cut by TevCas9;

[0181] II. Linear double-stranded DNA of different lengths, which contains homologous regions flanking the site targeted / cut by TevCas9;

[0182] III. Double-stranded DNA of the same length, which is cut by a nuclease and also contains DNA ends complementary to the DNA ends cut by TevCas9;

[0183] IV. Circular double-stranded DNA, which contains homologous regions flanking the site targeted / cut by TevCas9; and

[0184] V. Circular double-stranded DNA, which contains the I-TevI target site and the Cas9 target site, where the product cut from the double-stranded DNA contains ends complementary to the ends cut by TevCas9.

[0185] Example 4: Method for assembling lipid-encapsulated TevCas9 and transfecting cells

[0186] For ex vivo cell transfection: To assemble lipid-encapsulated TevCas9, lipid nanoparticles were mixed with TevCas9 at a molar ratio of 2000:1 in Dulbecco's Modified Eagle Medium (DMEM) (Sigma, St. Louis, Missouri, USA) and incubated at room temperature for 10 minutes. Cells were transfected with lipid-encapsulated TevCas9 at 8.7 x 10E-17 to 3.1 x 10E-17 moles per cell.

[0187] For in vivo cell transfection: To assemble lipid-encapsulated TevCas9, lipid nanoparticles were mixed with TevCas9 at a molar ratio of 2000:1 in phosphate-buffered saline and incubated at room temperature for 10 minutes. The molar ratio of lipid-encapsulated TevCas9 per cell for in vivo transfection needs to be determined.

[0188] Other embodiments

[0189] The nuclease may contain different combinations of the I-TevI domain, the linker domain, the Cas9 domain, or the guide RNA, as highlighted below.

[0190] Modifications of the I-TevI domain: Other versions of the I-TevI nuclease domain may contain different combinations of mutations to alter the site targeted by the I-TevI domain or the activity of the I-TevI domain, including mutations that alter the sequence recognized by I-TevI, such as K26 and / or C39. Other versions of the nuclease may replace the I-TevI domain with other GIY-YIG nuclease domains such as I-BmoI, Eco29kI, etc. Other versions do not contain Met due to processing when expressed in E. coli 1 .

[0191] Modifications of the linker domain: The linker domain may contain one or more of the following to alter the binding specificity or activity of TevCas9, including: a). The I-TevI linker domain, which contains one or more mutations of the amino acids T95, V117, K135, Q158, or N140; b). The linker may contain various combinations of the amino acids shown in SEQ ID NOs: 9-12

[0192] Modifications of the Cas9 domain: Other versions of the Cas9 domain may contain the following: a). A version of the saCas9 domain that contains the D10E mutation (SEQ ID NO: 14); b). A version of the saCas9 domain that cuts the target DNA on one strand of the target DNA, such as the H557A mutation (SEQ ID NO: 17); c). A version of the saCas9 domain that binds the target DNA but does not cut it, such as mutations at both the D10A and H557A mutations (SEQ ID NO: 18); d). A version of the previously described spCas9 EQR mutant that contains the mutations D1135E, R1335Q, and T1337R in combination with the D10E mutation (SEQ ID NO: 19); and e). A version of the previously described spCas9 EQR mutant that contains the mutations D1135E, R1335Q, and T1337R in combination with the D10E mutation, and a mutation that cuts the target DNA on one strand of the target DNA, such as the H840A mutation (SEQ ID NO: 20). Other versions of the saCas9 domain do not contain Met 1 .

[0193] Other versions can replace the Cas9 domain with other nucleases or DNA-binding domains, such as: a). Meganucleases, such as the LAGLIDADG family, His-Cys Box, H-N-H, PD-(D / E)xK, Vsr-like, etc.; b). Zinc finger nucleases; c). Other CRISPR proteins, such as scCas9, fnCas9, cjCas9, Cpf1, Cas12a, Cas13a, Cas3, etc.; and d). Other DNA-binding domains, such as zinc finger motifs, TALE activation domains, etc.

[0194] Modifications of the guide RNA: a). Other versions of the guide RNA can target the same DNA region in the CFTR gene or the EGFR gene, but contain different sequences to account for gene polymorphisms in the population; b). Other versions of the guide RNA can target different sequences in the CFTR gene or the EGFR gene; c). Other versions of the guide RNA can target other sequences in the genome to retarget the nuclease to other clinically relevant targets; d). Other versions of the guide RNA can contain bridged nucleic acids ("BNA") to enhance target site specificity; and e). Other versions can contain a mixture of guide RNAs to target multiple sequences within the same gene.

[0195] Modifications of the lipid nanoparticles: a). Other versions of lipid nanoparticles 1, 2, or 3 can have different ratios of each lipid component; b). Other versions of lipid nanoparticles can have different average diameters; c). Other versions of lipid nanoparticles can include different cationic or neutral lipids; d). Other versions of lipid nanoparticles can include peptides that target specific cell types; e). Other versions of lipid nanoparticles can include compounds that bind to DNA, such as GL67 (N 4 -cholesteryl-spermine); f). The lipid nanoparticles can be lyophilized to improve stability; and g). The lipid nanoparticles can be resuspended in solutions other than phosphate buffered saline, such as sterile isotonic saline, water for injection, etc.

[0196] Modifications of the composition of the donor DNA: a). Other versions of linear double-stranded donor DNA can contain longer single-stranded DNA regions complementary to the target sequence; and b). Other versions of circular double-stranded DNA can contain other DNA sequences designed to increase the rate of homologous directed repair.

[0197] Variants of methods for assembling lipid-encapsulated nucleases and transfecting cells: a). Other versions of lipid-encapsulated nucleases may contain different molar ratios of lipid nanoparticles to nuclease; b). Media other than DMEM or phosphate-buffered saline may be used for the incubation step; c). The nuclease and lipid nanoparticles may be incubated for less than or more than 10 minutes; and d). Other molar amounts of nuclease per cell may be used in the transfection reaction.

[0198] Variants of methods for manufacturing nuclease: a). Other E. coli expression strains may be used, such as LS5218 (Escherichia coli Genetic Stock Center - Yale University, New Haven, Connecticut, USA) or BL21-DE3 (New England Biolabs, Ipswich, Massachusetts, USA) b). Buffer 1 or 2 may contain different concentrations of imidazole, sodium chloride, sodium phosphate (dibasic), or tris hydrochloride and be buffered to different pHs; c). Other processing steps may be used, such as cation or anion exchange chromatography; d). The nuclease may be dialyzed into a solution other than phosphate-buffered saline, such as sterile isotonic saline, water for injection, etc.; e). The nuclease may be lyophilized to increase stability; f). Guide RNA may be co-expressed from a pACYC-Duet1 expression vector (EMD Millipore, Burlington, Massachusetts, USA). The DNA coding sequence for synthetic guide RNA (Integrated DNA Technology, Coralville, Iowa, USA) is digested with a restriction endonuclease and inserted into a similarly digested second expression site in the pACYC-Duet1 expression vector; and g). Guide RNA may be synthesized from double-stranded DNA by transcription using the T7 RNA polymerase HiScribe kit (New England Biolabs #E2040S, Ipswich, Massachusetts, USA) and purified using the RNA Cleanup kit (New England Biolabs #T2030L, Ipswich, Massachusetts, USA).

[0199] Testing

[0200] Example 1: Method for demonstrating correction of CFTRδF508 and functionality of CFTR protein in model cell lines

[0201] Method

[0202] A culture of immortalized epithelial cells homozygous for the CFTRδF508 mutation, such as the CuFi-1 cell line ( CRL-4013 TMThe American Type Culture Collection, Manassas, Virginia, USA), was treated with a series of concentrations of lipid-encapsulated TevCas9 and donor DNA (Specific Biologics, Toronto, Ontario, Canada) in a pharmaceutical formulation targeting the CFTR ΔF508 mutation. Appropriate control cell lines were also used, such as NuLi-1 homozygous for wild-type CFTR( CRL-4011 TM , American Type Culture Collection, Manassas, Virginia, USA) immortalized epithelial cells.

[0203] Measured by T7 endonuclease I assay( Mutation Detection kit, New England Biolabs #E3321, Ipswich, Massachusetts, USA), restriction endonuclease digestion of PCR-amplified target sites (New England Biolabs, Ipswich, Massachusetts, USA), deep gene sequencing using the Illumina MiSeq system and barcode primers flanking the target site (Illumina, San Diego, California, USA) or other suitable methods to measure the ratio of cells with corrected CFTR ΔF508 to uncorrected cells. Measured the effect of TevCas9 treatment in control cell lines (e.g., NuLi-1( CRL-4011 TM , American Type Culture Collection, Manassas, Virginia, USA). In the treated CuFi-1 cultures( CRL-4013 TM , American Type Culture Collection, Manassas, Virginia, USA) and mock-treated CuFi-1 cultures( CRL-4013 TM , American Type Culture Collection, Manassas, Virginia, USA), in the presence of a chloride ion gradient, short-circuit current measurements were used in an Ussing Chamber (Warner Instruments, Hamden, Connecticut, USA) to measure CFTR functionality. Also measured the effect of TevCas9 treatment in control cell lines (e.g., NuLi-1).

[0204] To demonstrate disruption of one or more EGFR exon 19 deletion mutations and EGFR expression and activity in a model cell line, cultures of immortalized epithelial cells expressing one or more EGFR exon 19 deletion mutations, such as the HCC827 cell line ( CRL-2868 TM , American Type Culture Collection, Manassas, Virginia, USA), were treated with a series of concentrations of lipid-encapsulated TevCas9 (Specific Biologics, Toronto, Ontario, Canada) in a pharmaceutical formulation in phosphate-buffered saline, sterile isotonic saline, or water for injection targeting the EGFR exon 19 deletion. Appropriate control cell lines were used, such as NuLi-1 homozygous for wild-type EGFR ( CRL-4011 TM , American Type Culture Collection, Manassas, Virginia) or an immortalized epithelial cell line.

[0205] The proportion of cells in which the EGFR exon 19 deletion was disrupted relative to uncorrected cells was measured by T7 endonuclease I assay ( Mutation Detection kit, New England Biolabs #E3321, Ipswich, Massachusetts, USA), restriction endonuclease digestion of PCR-amplified target sites (New England Biolabs, Ipswich, Massachusetts, USA), deep gene sequencing using the Illumina MiSeq system and barcoded primers flanking the target site (Illumina, San Diego, California, USA), or other suitable methods. The effect of TevCas9 treatment in control cell lines, such as NuLi-1 ( CRL-4011 TM , American Type Culture Collection, Manassas, Virginia, USA) was also measured. EGFR protein expression and activity were measured using an enzyme-linked immunosorbent assay (ELISA) (Sigma, St. Louis, Missouri, USA), which detected phosphorylated (i.e., activated), unphosphorylated, and total EGFR protein in treated HCC827 cultures ( CRL-2868 TM , American Type Culture Collection, Manassas, Virginia, USA) and mock-treated HCC827 cultures. The effect of TevCas9 treatment in control cell lines, such as NuLi-1 ( CRL-4011 TM,American Type Culture Collection, Manassas, Virginia, USA)).

[0206] Example 1: Animal model test for planning to show efficacy and determine dose-limiting toxicity

[0207] In an exemplary method of demonstrating the correction of CFTRδF508 and / or cystic fibrosis symptoms treated with lipid-encapsulated TevCas9 in an animal model (e.g., mouse, rat, minipig, or ferret), the lipid-encapsulated TevCas9 targeting CFTRδF508 in a pharmaceutical formulation of phosphate-buffered saline, sterile isotonic saline, or water for injection is delivered directly to the lungs by intubation or intranasal administration. The procedural time for each treatment is approximately 30 - 6000 seconds, depending on the animal model used.

[0208] In another method, the lipid-encapsulated TevCas9 in a pharmaceutical formulation targeting CFTRδF508 is nebulized using a commercial nebulizer ( (Trudell Medical, London, Ontario, Canada)) or PARI-LC (PARI USA, Midlothian, Virginia, USA)). After nebulization, the average size of the lipid nanoparticles is confirmed to be approximately 100 nM by dynamic light scattering (DLS) using a Zetasizer (Malvern Panalytical Ltd., Malvern, UK) or other suitable techniques known in the art. The composition and concentration of the lipid-encapsulated TevCas9 are confirmed using a MicroGram lipid assay kit (ProFoldin, Hudson, Massachusetts, USA) and the presence of a band of approximately 150 kDa on a Coomassie-stained SDS-polyacrylamide gel. For the measurement of the gene correction rate, a representative ovine (minipig) animal model (Exemplar Genetics, Sioux City, Iowa, USA) homozygous for the CFTRδF508 mutation is exposed to the lipid-encapsulated TevCas9 targeting CFTRδF508 and a suitable control by mouth, nose, or directly to the lungs. The general maintenance of these animals includes breeding and parturition; age-appropriate biosafety housing; good nutrition; basic vaccination and veterinary care; and documentation in compliance with animal welfare guidelines. The maintenance of these CFTRδF508-specific animals may include one or more of the following: surgical treatment of intestinal obstruction; pancreatic enzyme replacement therapy; vitamins and H2 blockers; and / or proton pump inhibitors to improve gastric acid control. The minipigs are treated with a series of concentrations of lipid-encapsulated TevCas9 predicted to be effective for acute toxicity studies for 2 days to 4 weeks and for chronic toxicity studies for up to 24 months based on the above model cell line studies.

[0209] Monitor the overall health of the animals after treatment to evaluate any treatment-related adverse events, such as changes in behavior, body weight, or food consumption; immune response; changes in cardiovascular health; death, etc. Other post-treatment efficacy measurements may include:

[0210] I. Forced exhalation, such as forced expiratory volume after treatment for each animal (or other suitable method);

[0211] II. The overall survival rate of each animal relative to the control;

[0212] III. Other pulmonary function measurements (e.g., using a mechanical ventilator that can perform general pulmonary function assessment); and

[0213] IV. Measuring in vivo mutations by tissue sampling and mutation detection methods such as polymerase chain reaction.

[0214] After treatment with lipid-encapsulated TevCas9, the animals are sacrificed and lung and tracheal tissues are harvested.

[0215] By T7 endonuclease I assay ( Mutation Detection kit, New England Biolabs #E3321, Ipswich, Massachusetts, USA), restriction endonuclease digestion of PCR-amplified target sites (New England Biolabs, Ipswich, Massachusetts, USA), deep gene sequencing using the Illumina MiSeq system and barcode primers flanking the target sites (Illumina, San Diego, California, USA) or other suitable methods to measure the ratio of cells with corrected CFTRδF508 to uncorrected cells.

[0216] In a method of demonstrating the disruption of one or more EGFR exon 19 deletion mutations and / or symptoms of non-small cell lung cancer (NSCLC) by treatment with TevCas9 in an animal model, lipid-encapsulated TevCas9 targeting the EGFR exon 19 deletion mutation in a pharmaceutical formulation in phosphate-buffered saline, sterile isotonic saline, or water for injection is delivered directly to the lungs by mouth, nose, or directly to the lung. The procedural time for each treatment is approximately 30 - 6000 seconds, depending on the animal model used.

[0217] In another method, lipid-encapsulated TevCas9 targeting the EGFR exon 19 deletion mutation in a pharmaceutical formulation is used with a commercial nebulizer (( (Trudell Medical, London, Ontario, Canada) or PARI-LC (PARI USA, Midlothian, Virginia, USA)) Atomization. After atomization, the average size of the lipid nanoparticles was confirmed to be approximately 100 nM by dynamic light scattering (DLS) using a Zetasizer (Malvern Panalytical Ltd., Malvern, UK) or other suitable techniques known in the art. The composition and concentration of the lipid-encapsulated TevCas9 nanoparticles were confirmed using a MicroGram Lipid Assay Kit (ProFoldin, Hudson, Massachusetts, USA) and the presence of a band of approximately 150 kDa on a Coomassie-stained SDS-polyacrylamide gel. To measure the gene disruption rate, a representative murine (mouse) animal model homozygous for one or more EGFR exon 19 deletion mutations was exposed to lipid-encapsulated TevCas9 targeting EGFR exon 19 deletion via the nose, mouth, or directly to the lung. The mice were treated with a series of concentrations of TevCas9 predicted to be effective for acute toxicity studies for 2 days to 4 weeks and for chronic toxicity studies for up to 24 months based on model cell line studies.

[0218] After treatment, the overall health of the animals was monitored to assess any treatment-related adverse events such as changes in behavior, body weight, or food consumption; immune response; changes in cardiovascular health; death, etc. Other post-treatment efficacy measurements may include:

[0219] I. Quantifying EGFR-activated protein by positron emission tomography (PET) using an EGFR mutation tracer;

[0220] II. The overall survival rate of each animal relative to the control; and

[0221] III. Measuring tumor formation / reduction in each animal over time.

[0222] In vivo mutations were measured by tissue sampling and mutation detection methods such as EGFR Mutation Test Version 2 (Roche Diagnostics, Risch-Rotkreuz, Switzerland). After treatment with atomized lipid-encapsulated TevCas9, the animals were sacrificed and lung and tracheal tissues were harvested.

[0223] By T7 endonuclease I assay ( Mutation Detection kits (New England Biolabs, E3321, Ipswich, Massachusetts, USA), restriction endonuclease digestion of PCR-amplified target sites (New England Biolabs, Ipswich, Massachusetts, USA), deep gene sequencing using the Illumina MiSeq system and barcode primers flanking the target sites (Illumina, San Diego, California, USA) or other suitable methods are used to measure the proportion of cells with disrupted EGFR exon 19 deletion mutations relative to non-disrupted cells. Enzyme-linked immunosorbent assay (ELISA) (Sigma, St. Louis, Missouri, USA) is used to measure EGFR protein expression and activity in harvested tissue cells, which detects phosphorylated (i.e., activated), unphosphorylated, and total EGFR proteins. Determining the dose-limiting toxicity capable of conducting the first-in-human clinical study is based on the predicted effective dose from the above animal model studies. Lipid-encapsulated TevCas9 at a series of concentrations (e.g., milligrams per kilogram body weight) is nebulized and delivered to a suitable animal model for toxicology studies, such as cynomolgus monkeys or other non-human primates. Monitor the overall health status of the animals for any treatment-related adverse events, such as changes in behavior, body weight, or food consumption; immune responses; changes in cardiovascular health; death, etc. Other measurements that can be used to measure efficacy in the study include those described above.

[0224] Therapeutic effect

[0225] The novel chimeric nucleases of the present invention have been deliberately designed to modify the DNA of lung epithelial cells to treat monogenic diseases, although they are capable of acting in vivo or ex vivo in other cell types or other organisms such as bacteria, yeast, insects, plants, or cells of other mammals to treat monogenic or polygenic and infectious diseases.

[0226] Example 1: Method for targeted insertion or replacement of DNA sequences in all or part of the human cell genome

[0227] Figure 2 Illustrates the mechanism of action of cellular uptake of the novel chimeric nucleases of the present invention. As Figure 2 shown in A, by in vivo or ex vivo administration, cells 20 or a plurality of cells 20 are exposed to novel lipid-encapsulated nuclease particles 21 containing TevCas9 25. As Figure 2 shown in B of, the lipid-encapsulated nuclease particles 21 are endocytosed into cells 20, and endosomes 22 undergo a maturation process in the cytosol and are targeted for degradation ( Figure 2 shown in C of). In some cases, TevCas9 25 can escape from endosomes 22 and enter the cytosol ( Figure 2D). In eukaryotes, the nuclease (TevCas9) 25 targets the nucleus 23 of the cell 20 through one or more nuclear localization sequences (“NLS”). As Figure 2 depicted in E below, through its nuclear localization sequence, TevCas9 25 can enter the nucleus 23, and when in the nucleus 23, the TevCas9 nuclease 25 binds to and cleaves 26 the target genomic DNA 24 sequence.

[0228] Figure 3 illustrates the mechanism by which the TevCas9 nuclease cleaves DNA. Figure 3 A of below shows the key features of TevCas9 bound to its target genomic DNA sequence 24 prior to the cleavage reaction. The I-TevI domain 27 targets the I-TevI target sequence 29. The linker domain 30 connects the I-TevI domain 27 to the Cas9 domain 28 that targets the Cas9 target sequence 31. The gene mutation 32 is surrounded by or near the I-TevI target sequence 29 and the Cas9 target sequence 31. As Figure 3 shown in B of below, TevCas9 25 cleaves the target sequence, leaving a deletion product 34 of predictable size with non-complementary DNA ends 35, 36. Figure 3 C of below illustrates that in the presence of single-stranded donor DNA with homologous arms 37, the cell 20 can insert the donor DNA 37 sequence near the cleavage site through the homologous directed repair (HDR) pathway 38. Figure 3 D of below illustrates that in the presence of donor DNA 39 with DNA ends compatible with the DNA ends cleaved by TevCas9 25, the cell 20 can insert the donor DNA sequence 39 between the cleavage sites using the non-homologous end joining (NHEJ) pathway 40 by directed ligation. In the absence of donor DNA, the cell 20 can ligate the DNA ends through the NHEJ pathway 40 ( Figure 3 E of below).

[0229] Example 2: Treatment of cystic fibrosis

[0230] For the treatment of cystic fibrosis, the exogenous donor DNA contains the DNA sequence that repairs the CFTRδF508 mutation, involving a method of using exogenous donor DNA as a template to targetedly delete a defined length of DNA sequence in human somatic cells to stimulate homologous directed repair ( Figure 3 C of below).

[0231] Example 3: Treatment of non-small cell lung cancer

[0232] For use in the treatment of non-small cell lung cancer, a Cas9 domain version that cleaves only one strand of DNA (D10A or H557A mutation) or a nuclease-deficient version (D10A + H557A mutation) is used, and the targeted sequence is the EGFR exon 19 deletion mutation (SEQ ID NO: 2-4). However, in the present application, the nuclease does not contain exogenous donor DNA. In the absence of exogenous donor DNA, the cell can remove the DNA sequence between the two sites targeted by the nuclease by non-homologous end joining ( Figure 3 of E).

[0233] Alternatively, the inhalation route is a rapid and effective way to deliver drugs locally to the lungs and for systemic administration of certain reagents. Inhaled drug therapy is widely used to treat respiratory conditions such as asthma and chronic obstructive pulmonary disease (COPD). Research is ongoing to develop inhalation systems for the treatment of cystic fibrosis.

[0234] The following examples are not intended to limit the scope of the disclosure, but rather to provide illustrations of how to prepare and use the compounds disclosed herein. Many other embodiments of the disclosure will be apparent to those skilled in the art.

[0235] A nebulizer is a device that delivers drugs to the lungs in the form of an aerosolized vapor. Nebulizers are commonly used to treat respiratory diseases such as asthma and COPD, for example, the nebulization of corticosteroids, although nebulization has also been used to treat and prevent lung infections such as (Insmed Incorporated, Bridgewater, New Jersey, USA).

[0236] The nebulizer may require some procedures to prepare the liquid for nebulization. The drug is usually placed in a cup in the nebulizer chamber in liquid form. After loading, the device is turned on, and compressed air is generated to convert the liquid into vapor in the nebulizer chamber. The patient places the mouthpiece of the nebulizer chamber in the mouth and takes a deep, forceful breath and holds the breath for 5-10 seconds to ensure that the drug reaches the lower part of the lungs. There are various such devices. Many modern nebulizers are breath-actuated and rely on the force of the patient's inhalation to draw the nebulized liquid out of the device, thus ensuring that the drug is delivered only to the patient and not to the surrounding environment. This also ensures consistency in delivering the full dose of the drug to the patient.

[0237] The use of nebulizers is well known, and nebulizers are commercially available from multiple sources such as (Trudell Medical, London, Ontario, Canada) or PARI-LC (PARI USA, Midlothian, Virginia, USA). In an example of the present invention, a nebulizer is used to deliver a novel lipid-encapsulated chimeric nuclease of the present application, which comprises a modified I-TevI nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9, to lung epithelial tissue. A sterile liquid version of a therapeutic agent of interest is loaded into the nebulization chamber and subsequently nebulized and inhaled into the lungs by the patient via deep breathing.

[0238] Some advantages of using a nebulizer compared to oral or intravenous administration are: less drug may be required compared to oral or intravenous administration; onset of action may be faster by inhalation compared to the oral route; adverse reactions may be less severe as the drug is delivered locally to the lung tissue where the disease itself appears; inhaled drug therapy is painless and relatively comfortable for the patient, which encourages compliance.

[0239] No non-invasive route of administration can provide the speed of action that inhaled drugs can provide. One of the advantages of inhaled drugs is that they are absorbed faster than subcutaneously injected molecules and provide a more direct physiological response. Small or large molecules, especially hydrophobic molecules, can be absorbed within seconds of inhalation and can therefore be used to treat a variety of symptoms that occur suddenly or require long-term administration. Pain, panic, anxiety, nausea, cardiovascular crisis, bronchoconstriction, sleep induction, spasm, Parkinson’s lock-up, and hot flashes are some of the rapidly developing conditions that inhaled drugs can address.

[0240] Most protein-based drug products have some water solubility and are rapidly and effectively absorbed from the lungs. Those that are more hydrophobic are even more rapidly absorbed within seconds to minutes. Those that are more hydrophilic are absorbed within minutes to tens of minutes. In an example of the present invention, one vial is aseptically filled with a therapeutic dose of hydrophobic lipid nanoparticles, and another vial is aseptically filled with a therapeutic dose of a chimeric nuclease comprising a modified I-TevI nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9, which is water soluble and hydrophilic, for delivery to the lungs at a therapeutic dose. The dose of each of the lipid nanoparticles and the chimeric nuclease can range from 1 to 1000 milligrams, preferably about 5 to 200 milligrams. The claimed lipid-encapsulated chimeric nuclease comprising a modified I-TevI nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9 can be absorbed by lung cells within a few hours, and complete cleavage on a DNA substrate has been observed in vitro within 2 hours. Other nebulized therapies have been delivered daily. Thus, nebulized administration of the lipid-encapsulated chimeric nuclease can be administered once daily or less frequently, depending on its efficacy based on each patient. The chimeric nuclease is produced by BioVectra Inc. (Charlottetown, PE, Canada), and the vials are aseptically filled by Dalton Pharma Services (Mississauga, Ontario, Canada). The lipid nanoparticles are manufactured by Transferra Nanosciences Inc. (Burnaby, BC, Canada) and the vials are aseptically filled.

[0241] The dose of any disclosed composition will vary according to the symptoms, age, and weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the composition. Any of the present formulations can be administered in a single dose or divided doses. The dose of the composition can be readily determined by techniques known to those skilled in the art or as taught herein.

[0242] In certain embodiments, depending on the weight of the patient, the dose of the present compound is generally in the range of about 1 to 1000 milligrams, particularly in the range of about 5 to 200 milligrams.

[0243] For any particular composition of the present disclosure, it may be necessary to determine an effective dose or amount and any possible effect on the timing of administration of the formulation. This can be accomplished by conventional experimentation as described herein, using one or more groups of animals (preferably at least 5 animals per group), or if appropriate, in human trials. The effectiveness of any of the present compositions and methods of treatment or prevention can be evaluated by administering the composition and assessing the effect of administration by measuring one or more applicable indices and comparing the post-treatment values of these indices with the pre-treatment values of the same indices.

[0244] The exact administration time and amount of any particular composition of the present invention that will produce the most effective treatment in a given patient will depend on the activity, pharmacokinetics, and bioavailability of the composition, the physiological condition of the patient (including age, gender, disease type and stage, general physical condition, response to a given dose and type of drug), the route of administration, and the like. The guidelines presented herein can be used to optimize treatment, such as determining the optimal administration time and / or amount, which will only require routine experimentation consisting of monitoring the subject and adjusting the dose and / or time. When a subject is being treated, the health of the patient can be monitored by measuring one or more relevant parameters at predetermined times during the treatment. The treatment, including the composition, amount, administration time, and formulation, can be optimized based on the results of such monitoring. By measuring the same parameters, the patient can be periodically re-evaluated to determine the degree of improvement. Adjustments can be made to the amount of the composition of the present invention administered and possibly the administration time based on these re-evaluations.

[0245] Treatment can be initiated with a smaller dose than the optimal dose of the compound. Thereafter, the dose can be increased in small increments until the optimal therapeutic effect is achieved.

[0246] The use of the composition of the present invention can reduce the required dose of any individual reagent contained in the composition because the onset and duration of action of different reagents can be complementary. The therapeutic efficacy of the composition of the present invention can be determined by standard pharmacological procedures in cell cultures or experimental animals, such as those used to determine LD 50 and ED 50 .

[0247] Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for humans. The dose of any composition of the present invention is preferably within a concentration range that includes ED 50 and has little or no toxicity. The dose can vary within this range depending on the dosage form employed and the route of administration utilized. For the compositions of the present disclosure, the therapeutically effective dose can initially be estimated from cell culture assays.

[0248] Formulations

[0249] The pharmaceutical compositions of the present disclosure can be administered in a variety of ways depending on their intended use, as is well known in the art. By way of example, the compositions of the present disclosure will be administered by nebulization. Alternatively, the formulations disclosed herein can be administered intravenously, subcutaneously, or intramuscularly. These formulations can be prepared by conventional means and, if desired, the composition can be mixed with any conventional additives such as excipients, solubilizing agents, suspending aids, emulsifying agents, or preservatives. The disclosed excipients can be used for more than one function. By way of example, a solubilizing agent can also be a suspending aid, emulsifying agent, preservative, etc.

[0250] The pharmaceutical compositions can conveniently be presented in unit dosage form and can be prepared by any methods well-known in the pharmaceutical art. The amount of the compositions which can be combined with a carrier material to produce a single dosage will vary depending on the subject being treated and the particular mode of administration.

[0251] The methods of preparing these pharmaceutical compositions include the step of bringing the compositions of the present disclosure into association with a carrier and, optionally, one or more accessory ingredients. Generally, the pharmaceutical compositions are prepared by uniformly and intimately bringing the reagents into association with a liquid carrier.

[0252] It should be understood that the disclosed compositions can include the lyophilized or freeze-dried compounds disclosed herein. For example, disclosed herein are compositions in the form of crystalline and / or amorphous powders of the disclosed compounds. Such forms can be reconstituted for use, for example, as aqueous compositions.

[0253] Liquid dosage forms for injection include pharmaceutically acceptable solutions, emulsions, microemulsions, solutions and suspensions. In addition to the present compositions, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, glycerol, tetrahydrofurfuryl alcohol, and fatty acid esters of sorbitan, cyclodextrin, albumin, hyaluronic acid, chitosan and mixtures thereof. Polyethylene glycol (PEG) can be used to obtain desired properties in solubility, stability, half-life and other pharmaceutically advantageous properties. Representative examples of stabilizing components include polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose and combinations thereof. Other excipients that can be used such as solution binders or antioxidants include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose sodium, crospovidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C and xylitol. Generally, aqueous aerosols are prepared by formulating an aqueous solution or suspension of the present compositions together with a conventional pharmaceutically acceptable carrier and stabilizer. The carrier and stabilizer vary with the particular requirements of the present compositions, but generally include nonionic surfactants (Tweens, pluronics or polyethylene glycol), innocuous proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

[0254] It should be noted that the excipients given as examples may have more than one function. For example, a solubilizer can also be a suspending aid, an emulsifier, a preservative, etc.

[0255] Examples of suitable aqueous and non-aqueous carriers for the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate and cyclodextrin. For example, appropriate fluidity can be maintained by using coating materials such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants.

[0256] The compositions of the present invention are generally sterile, storage-stable and pharmaceutically acceptable aqueous solutions that are easy to administer and do not require reconstitution before administration. The compositions of the present invention are suitable for administration to a subject, which means that they are pharmaceutically acceptable, non-toxic, do not contain any components that would have an adverse effect on the biological effect of the chimeric nuclease, and have a pH close to physiological conditions, which avoids inhalation and / or injection site reactions. For example, the compositions of the present invention do not contain any cells.

[0257] The compositions are generally stored in sealed containers, vials or cartridges that are generally suitable for long-term storage. "Suitable for long-term storage" means that the vial, container or cartridge will not allow the components of the compositions of the present invention to escape or for external components, such as microorganisms, to enter when maintained at 25 °C for at least 3 months. The compositions of the present invention are preferably administered by nebulization, typically breath-actuated nebulization.

[0258] The compositions of the present invention can also be administered by injection as described herein.

[0259] The compositions of the present invention can be administered alone or in combination with additional therapeutic agents, such as antiviral agents, antimicrobial agents, chemotherapeutic agents and immunotherapies.

[0260] As used herein, a vial can also comprise two containers, where one container contains the chimeric nuclease or lipid particles as described herein in the form of a lyophilized powder as described below, and the second container contains a liquid for the reconstitution of the lyophilized powder. The contents of the two containers can be mixed before administration.

[0261] As described above, the compositions of the present invention can be administered by nebulization. Suitable volumes of the compositions of the present invention for nebulization include from about 0.5 to about 1 ml, from about 1 to about 2 ml, from about 2 to about 10 ml or from about 10 to about 20 ml.

[0262] In the composition of the present invention, the concentration of the chimeric nuclease is about 0.1 mg / ml to about 10.0 mg / ml, about 10.0 mg / ml to about 100.0 mg / ml, about 30.0 mg / ml to about 300.0 mg / ml, about 500 mg / ml to about 2000 mg / ml, and about 2.0 mg / ml.

[0263] In the composition of the present invention, the concentration of the lipid nanoparticles is about 0.1 mg / ml to about 10.0 mg / ml, about 10.0 mg / ml to about 100.0 mg / ml, about 30.0 mg / ml to about 300.0 mg / ml, about 500 mg / ml to about 2000 mg / ml, and about 2.0 mg / ml.

[0264] SEQ ID NO 1

[0265]

[0266]

[0267] Length: 4,443

[0268] Type: DNA

[0269] Organism: Homo sapiens

[0270] Features: Cystic fibrosis transmembrane conductance regulator (cFTR) δF508 DNA coding sequence

[0271] Other information: Nucleotides deleted compared to wild-type CFTR are shown as ("-"). The sequence targeted by TevCas9 is highlighted. The I-Tevl site is underlined, the Cas9 guide RNA sequence is double-underlined, and the PAM sequence is in lowercase.

[0272] SEQ ID NO 2

[0273]

[0274] Length: 132

[0275] Type: DNA

[0276] Organism: Homo sapiens

[0277] Features: Epidermal growth factor receptor (EGFR) exon 19 C.2235-2249 deletion sequence

[0278] Other information: Nucleotides deleted compared to wild-type EGFR are shown as ("-"). The sequence targeted by TevCas9 is highlighted. The I-Tevl site is underlined, the Cas9 guide RNA sequence is double-underlined, and the PAM sequence is in lowercase. Intron sequences are in bold italics.

[0279] SEQ ID NO 3

[0280]

[0281] Length: 132

[0282] Type: DNA

[0283] Organism: Homo sapiens

[0284] Feature: Epidermal growth factor receptor (EGFR) exon 19 C.2236_2250 deletion sequence

[0285] Other information: Nucleotides deleted compared to wild-type EGFR are shown as ("-"). The sequence targeted by TevCas9 is highlighted.

[0286] The I-Tevl site is underlined, the Cas9 guide RNA sequence is double-underlined, and the PAM sequence is in lowercase. Intron sequences are in bold italics.

[0287] SEQ ID NO4

[0288]

[0289] Length: 132

[0290] Type: DNA

[0291] Organism: Homo sapiens

[0292] Feature: Epidermal growth factor receptor (EGFR) exon 19 C.22372251 deletion sequence

[0293] Other information: Nucleotides deleted compared to wild-type EGFR are shown as ("-"). The sequence targeted by TevCas9 is highlighted.

[0294] The I-Tevl site is underlined, the Cas9 guide RNA sequence is double-underlined, and the PAM sequence is in lowercase. Intron sequences are in bold italics.

[0295] SEQ ID NO 5

[0296]

[0297] Length: 147

[0298] Type: DNA

[0299] Organism: Homo sapiens

[0300] FEATURE: Wild-type sequence of exon 19 of epidermal growth factor receptor (EGFR)

[0301] Other information: The sequence targeted by TevCas9 is highlighted. The I-Tevl site is underlined, the Cas9 guide RNA sequence is double-underlined, and the PAM sequence is in lowercase. Note: The I-Tevl and Cas9 target sites are not properly spaced, so I-Tevl should not bind or cleave its target site. The intron sequence is in bold italic.

[0302] SEQ ID NO 6

[0303]

[0304] Length: 93

[0305] Type: Amino acid

[0306] Organism: Enterobacteriophage T4

[0307] Feature: I-TEVI domain

[0308] Other information:

[0309] SEQ ID NO 7

[0310]

[0311] Length: 83

[0312] Type: Amino acid

[0313] Organism: Artificial

[0314] Feature: Linker domain

[0315] Other information: V117F; mutation is underlined

[0316] SEQ ID NO8

[0317]

[0318] Length: 83

[0319] Type: Amino acid

[0320] Organism: Artificial

[0321] Feature: Linker domain

[0322] Other information: K135R / N140S; mutations underlined

[0323] SEQ ID NO 9

[0324]

[0325] Length: 83

[0326] Type: Amino acid

[0327] Organism: Artificial

[0328] Feature: Linker domain

[0329] Other information: V117F / K135R / N140S; mutations underlined

[0330] SEQ ID NO 10

[0331]

[0332] Length: 86

[0333] Type: Amino acid

[0334] Organism: Artificial

[0335] Feature: Linker domain variant

[0336] Other information:

[0337] SEQ ID NO 11

[0338]

[0339] Length: 87

[0340] Type: Amino acid

[0341] Organism: Artificial

[0342] Feature: Linker domain variant

[0343] Other information:

[0344] SEQ ID NO 12

[0345]

[0346] Length: 95

[0347] Type: Amino acid

[0348] Organism: Artificial

[0349] Feature: Linker domain variant

[0350] Other information:

[0351] SEQ ID NO 13

[0352]

[0353] Length: 1,053

[0354] Type: Amino acid

[0355] Organism: Staphylococcus aureus

[0356] Features:

[0357] Other information:

[0358] SEQ ID NO 14

[0359]

[0360] Length: 1,053

[0361] Type: Amino acid

[0362] Organism: Artificial

[0363] Features: SACAS9 D10E mutation

[0364] Other information: Mutation underlined

[0365] SEQ ID NO 15

[0366]

[0367] Length: 101

[0368] Type: RNA

[0369] Organism: Artificial

[0370] Features: Target CFTR gene

[0371] Other information:

[0372] SEQ ID NO 16

[0373]

[0374] Length: 101

[0375] Type: RNA

[0376] Organism: Artificial

[0377] Features: Target EGFR gene

[0378] Other information:

[0379] SEQ ID NO 17

[0380]

[0381] Length: 1,053

[0382] Type: Amino acid

[0383] Organism: Artificial

[0384] Features: SACAS9 D10E + H557A mutation

[0385] Other information: Mutations are underlined

[0386] SEQ ID NO 18

[0387]

[0388]

[0389] Length: 1,053

[0390] Type: Amino acid

[0391] Organism: Artificial

[0392] Features: SACAS9 D10A + H557A mutation

[0393] Other information: Mutations are underlined

[0394] SEQ ID NO 19

[0395]

[0396] Length: 1,368

[0397] Type: Amino acid

[0398] Organism: Artificial

[0399] Features: SPCAS9 D10E, D1135E, R1335Q, T1337R mutations

[0400] Other information: Mutations are underlined

[0401] SEQ ID NO 20

[0402]

[0403] Length: 1,368

[0404] Type: Amino acid

[0405] Organism: Artificial

[0406] Features: SPCAS9 D10E, H840A, D1135E, R1335Q, T1337R mutations

[0407] Other information: Mutations are underlined

[0408] SEQ ID NO 21

[0409]

[0410] Length: 101

[0411] Type: RNA

[0412] Organism: Artificial Feature: Target CFTR δF508 gene

[0413] Other information:.

Claims

1. A polypeptide, said polypeptide comprising the complete amino acid sequence of SEQ ID NO: 14 or a fragment thereof, provided that said fragment contains Glu 10 mutation.

2. The polypeptide according to claim 1, wherein, The polypeptide is a modified RNA-guided nuclease Staphylococcus aureus Cas9.

3. A chimeric nuclease comprising a modified I-Tevl nuclease domain, a linker, an RNA-guided nuclease Staphylococcus aureus Cas9, and a guide RNA.

4. A pharmaceutically acceptable formulation comprising the chimeric nuclease according to claim 3, a lipid nanoparticle, and a pharmaceutically acceptable carrier thereof.

5. The preparation according to claim 4, wherein, The lipid nanoparticle further comprises exogenous donor DNA.

6. The formulation according to claim 5, further comprising a guide RNA, wherein the guide RNA targets the Cas9 domain to genomic DNA.

7. A method of editing genomic DNA, the method comprising the step of administering the chimeric nuclease according to claim 3 to a cell or an organism.

8. The method according to claim 7, wherein The method does not use a viral vector.

9. The method according to claim 8, wherein, The administration of the chimeric nuclease is carried out in vivo using a controlled dose.

10. A method of deleting a DNA molecule of a defined length, the method comprising the step of delivering the formulation according to claim 4 to a cell or an organism.

Images