Lipid-Encapsulated Double-Strand-Cleaving Endonucleases for DNA and Gene Editing

Abstract

A method for editing genes by administering chimeric nucleases to cells or organisms without the use of viral vectors.

Description

Technical Field

[0001] Array List This application contains an Array List submitted in ASCII format via EFS-Web, which is hereby incorporated by reference in its entirety. The ASCII copy created on September 17, 2018, is Sequence_Listing_ST25.txt and has a size of 77 KB.

Background Art

[0002] There are an estimated 5,000 to 10,000 monogenic diseases, defined as genetic conditions arising from mutations on a single gene. These diseases often manifest during childhood, leading to various conditions and sometimes death in young adulthood. Collectively, they are estimated to affect approximately 6% of people at some point in their lives. Diagnosis and treatment for these diseases are still often inadequate, and their care is mainly symptomatic, focusing on disease management and not addressing the underlying genetic deficiencies. There are also many additional diseases in which mutations in genes contribute to disease pathogenesis.

[0003] Gene editing is a designer nuclease-dependent gene therapy approach that recognizes and cleaves specific DNA sequences to introduce targeted modifications into the genome and then utilizes the natural cellular DNA repair pathways, namely non-homologous end joining (NHEJ) and homology-directed repair (HDR). Four nuclease families: meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided Cas9 (CRISPR-Cas9) nucleases associated with clustered regularly interspaced short palindromic repeats have been used in this regard. These can be designed to accurately introduce double-strand breaks at target gene loci of interest. Gene editing expands the potential to permanently modify genomic sequences of interest by enabling targeted disruption, insertion, cleavage, and correction in both ex vivo and in vivo settings. While these advantages are expected to revolutionize the field as a whole, current gene editing approaches are limited by the efficiency of modification, safety concerns related to nuclease specificity, and delivery of gene editing tools to target cell types.

[0004] The Cas9 (CRISPR-associated) protein, a component of the type II CRISPR system that constitutes the natural immune system of bacteria, has caused a paradigm shift in the field of genome editing due to its ease of use. Programming Cas9 to cleave a desired sequence simply involves changing the sequence of the Cas9-associated guide RNA to be complementary to the target site. The ease of programming Cas9 targeting contrasts with the more intensive protein modifications required for other reagents (zinc-finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs)). Along with proteins from type III CRISPR systems, Cas9 has been used for numerous genome editing applications in a wide range of organisms and is now entering the realm of therapeutic applications in humans.

[0005] Cystic fibrosis (CF) is an autosomal recessive genetic disorder that occurs due to mutations in the CFTR gene encoding an epithelial anion channel. The CFTR protein, a cystic fibrosis transmembrane conductance regulator, is found in a wide range of organs including the pancreas, kidneys, liver, lungs, gastrointestinal tract, and reproductive organs, thereby making CF a multi-organ disease. Mutations in CFTR result in suboptimal ion transport and water retention, leading to prominent clinical manifestations such as abnormal thickening of mucus in the lungs and pancreatic insufficiency. In the lungs, dysfunctional CFTR disrupts mucociliary clearance, making the organs prone to bacterial infection and inflammation, ultimately resulting in airway obstruction, respiratory failure, and premature death. CF remains the most common lethal genetic disorder in the white population, with an estimated 70,000 - 100,000 affected individuals worldwide, underscoring the real need for the development of better treatments.

[0006] One major challenge in the development of treatment strategies for CF is the wide variety of mutation types. The ΔF508 (deletion of phenylalanine at codon 508) mutation has a prevalence of >80% in CF patients and is the most common, but over 1,990 harmful CFTR mutations have been described. These mutations cause premature stop codons, abnormal splicing, inaccurate protein folding or transport to the cell surface, and dysfunctional CFTR with limited channel opening ability. Pharmacological interventions target some of these processes, with drug administration being therapeutic in some types of gating mutations, while the frequently occurring ΔF508 still requires more effective treatment. However, pharmaceutical advances in CF care do not address mutations resulting from abnormal splicing or premature stop codons; in these cases, gene editing may prove most advantageous.

[0007] Similarly, in populations in Europe and the United States, approximately 15% of patients with non-small cell lung cancer (NSCLC) have activating mutations in their tumors in the epidermal growth factor receptor (EGFR) gene.

[0008] Existing gene editing technologies such as CRISPR-Cas9 (and Cas9 fusions), meganucleases, zinc finger proteins, type II S restriction endonucleases (FokI and FokI fusions), and TALENs are limited in their ability to introduce specific-length gene deletions or accurately repair target genes in sufficient cell numbers to be meaningful as therapeutic agents for many genetic diseases. Furthermore, for highly programmable RNA-guided nucleases such as monomeric Cas9, experiments suggest that the specificity for binding, cleavage, and repair only at its target site is limited, raising concerns about potential harmful changes to the genomic DNA of cells that can inadvertently cause secondary diseases in patients. Finally, most nucleases are delivered in viral vectors. Viral vectors have the potential for existing immunity in many populations, post-treatment immunogenicity, and genotoxicity. Non-viral delivery methods for safely delivering nucleases to target cells and enabling controlled in vivo administration of nucleases do not currently exist.

[0009] The need for improvements to the above existing gene editing technologies to address the above concerns to make gene editing technologies more efficient and effective is not being met.

SUMMARY OF THE INVENTION

[0010] The present invention preferably lacks Met 1 and has Lys 26 (Lys 27 in the non-truncated version of I-TevI) and / or Cys 39 (Cys 40 ) modifications, a modified I-TevI nuclease domain, a linker, particularly SEQ ID NOs: 7-12 or a fragment thereof, and / or the following mutations Thr 95 (when referring to full-length I-TevI), Val 117 , Lys 135 , Gln 158 or Asn 140Those containing one or more of, which can be a wild-type or modified version, preferably its Glu 10 or Ala 557 A chimeric nuclease comprising a modified RNA-guided nuclease Staphylacoccus aureus Cas9 containing a mutation (the I-TevI polypeptide comprises the entire amino acid sequence of SEQ ID NO: 6 or a fragment thereof) and a guide RNA targeting the Cas9 domain, particularly SEQ ID NO: 15, 16, 21 or a fragment thereof, and the chimeric nuclease, cationic and / or neutral lipid nanoparticles, and optionally a DNA-binding compound, particularly GL67 (N 4 -cholesteryl-spermine), and a pharmaceutically acceptable carrier thereof, is targeted.

[0011] In a further embodiment of the invention, in the formulation the lipid nanoparticles can contain exogenous donor DNA.

[0012] Another embodiment of the invention is directed to a method for editing a gene by administering a chimeric nuclease to a cell or organism without using a viral vector by using controlled administration in vivo.

[0013] Another embodiment of the invention is directed to a method for deleting a defined length of a DNA molecule or replacing a selected sequence from a DNA molecule by delivering a chimeric nuclease to an entire organism in vivo or to isolated cells in culture ex vivo, wherein the cells are mammalian cells, bacteria, insect cells or plant cells.

[0014] In yet another embodiment, the novel chimeric nuclease targets two independent target sites on a selected DNA molecule, cleaves at either one or both of the target sites, and generates a fragment that is 30 - 36 nucleotides in length.

[0015] In a further example, the novel purified chimeric nuclease further comprises a guide RNA.

[0016] Another aspect of the present invention is the use of an extrusion step to generate particles approximately 100 nM in diameter containing an excipient, the excipient being 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 mixtures of these substances so that the novel chimeric nuclease can be administered to a patient using a nebulizer containing the formulation.

[0017] In a preferred embodiment, the present invention is directed to a method of treating a lung-related disease in a patient in need of treatment of the lung-related disease by administering a novel chimeric nuclease that modifies the DNA of lung epithelial cells, the chimeric nuclease replacing the CFTR delta F508 mutation from the CFTR gene to treat cystic fibrosis or cleaving the EGFR exon 19 deletion to treat non-small cell lung cancer.

[0018] In yet another embodiment, the present invention is directed 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 an Ala 10 , Ala 557 or Ala 580 mutation and targets the EGFR exon 19 deletion of the EGFR gene.

[0019] In a further embodiment, the guide RNA targets a specific CTFR gene sequence and excises a specific EGFR gene sequence containing the CFTR delta F508 mutation or the EGFR exon 19 deletion mutation.

[0020] The present invention also encompasses a modified donor DNA molecule selected from the group consisting of a linker containing SEQ ID NOs: 7-12 or a fragment thereof, a linear single-stranded DNA containing a homologous region adjacent to the site targeted and / or cleaved by a chimeric nuclease, a linear double-stranded DNA containing a homologous region adjacent to the site targeted and / or cleaved by a chimeric nuclease, a double-stranded DNA of the same length containing complementary DNA ends to that cleaved by a chimeric nuclease, a circular double-stranded DNA containing a homologous region adjacent to the site targeted and / or cleaved by a chimeric nuclease, and a circular double-stranded DNA containing an I-TevI target site and a Cas9 target site, wherein the product cleaved from the double-stranded DNA contains complementary ends to the ends cleaved by the chimeric nuclease.

[0021] In a further example, a chimeric nuclease comprising a modified GIY-YIG nuclease domain, a linker, and a modified RNA-guided nuclease Staphylacoccus aureus Cas9 or Streptococcus pyogenes Cas9 or Glu 10 mutation (SEQ ID NO: 19) and / or Ala 840 mutation and / or an EQR Streptococcus pyogenes Cas9 variant containing 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.

[0022] 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 CRISPR proteins selected from the group consisting of LAGLIDADG, His-Cys Box, H-N-H, PD-(D / E)xK, and Vsr-like meganucleases, Cas12a (Acidaminococcus Sp), Cas13a (Leptorichia shahii), and Cas3 (Streptococcus thermophilus), and DNA binding domains selected from the group consisting of zinc-finger motifs and TALE activator domains.

[0023] In yet a further example, the invention includes a modified RNA-guided nuclease Staphylacoccus aureus Cas9 and a guide RNA, wherein the guide RNA contains sequences that target genetic polymorphisms, different sequences in the CFTR or EGFR genes, sequences that retarget the nuclease, cross-linked nucleic acids, and / or a mixture of guide RNAs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

[0025] Definitions and Acronyms For convenience, certain terms used in this application, the examples, and the appended claims are summarized here. These definitions are to be read in light of the present disclosure and understood as would be by one 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.

[0026] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. The term "and / or" as used herein is defined as having the possibility of one or the other or both. For example, "A and / or B" provides a scenario of having only A or only B or a combination of A and B. When a claim indicates A and / or B and / or C, the composition may include A alone, B alone, C alone, A and B but not C, B and C but not A, A and C but not B, or all three of A, B, and C.

[0027] The term "bioavailable" is recognized in the art and refers to forms of the present disclosure that are incorporated into the subject, absorbed by the subject or patient to whom it is administered, or otherwise made physiologically available to the subject, where it or a portion of the amount administered is so made.

[0028] The term "exogenous donor DNA" as used herein refers to any sequence of DNA that is not wholly or partially the same as the original target DNA sequence.

[0029] The term "flexible linker" as used herein refers to the situation where the amino acid linker domain ensures the mobility of the I-TevI domain to enable recognition, binding, and cleavage of the target sequence under cellular physiological conditions (generally: pH~7.2, temperature~37°C, [K+]~140 mM, [Na+]~5 - 15 mM, [Cl-]~4 mM, [Ca++]~0.0001 mM) when the RNA-guided nuclease domain (Cas9) binds to the target DNA sequence. The length of the amino acid linker can affect how many nucleotides are preferred between the Cas9 target site and the I-TevI target site. Certain amino acids in the linker can also make no specific contact with the DNA sequence targeted by TevCas9. These linker-DNA contacts can affect the flexibility of the I-TevI domain. Amino acid substitutions in the linker domain can affect the ability of the linker domain to contact DNA.

[0030] The term "including", as used herein, is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[0031] The terms "inhalation administration", "inhaled", "inhaled by", "inhalation" or "inhalation therapy", as used interchangeably and as used herein, include the administration of a substantially uniform distribution of appropriately sized particles to the airway epithelium of the nose, central airways, peripheral side of the lung and / or alveolar region of the lung, or by intratracheal instillation. Such particles can be introduced to and / or generated for a patient using an appropriate device, preferably a nebulizer.

[0032] The terms "patient", "subject" or "host" treated by the method can mean either a human or a non-human animal. Non-human animals include companion animals (e.g., cats, dogs) and animals raised for food consumption (i.e., food animals), such as cows, pigs and chickens.

[0033] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is recognized in the art and involved in carrying or transporting any subject composition or its components from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense that it is compatible with the subject composition and its components and not harmful to the patient. Some examples of materials that can act 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) buffering agents, such as monobasic and dibasic phosphates, Tris / boric acid / EDTA, and Tris / acetic acid / EDTA; (8) pyrogen-free water; (9) isotonic saline; (10) Ringer's solution; (11) ethyl alcohol; (12) phosphate buffer; (13) polysorbate; (14) polyphosphate; and (15) other non-toxic compatible substances used in pharmaceutical formulations. The disclosed excipients can perform multiple functions. For example, a solubilizing agent can also be a suspending aid, emulsifying agent, preservative, etc.

[0034] In certain preferred embodiments, the pharmaceutically acceptable excipient is a crystalline bulking excipient. The term "crystalline bulking excipient" or "crystalline bulking agent" as used herein means an excipient that provides bulk and structure to the lyophilized cake. These crystalline bulking agents are inert and do not react with the protein or nucleic acid. Further, the crystalline bulking agent can crystallize in the lyophilized state. Examples of suitable crystalline bulking agents include hydrophilic excipients such as water-soluble polymers; sugars such as mannitol, sorbitol, xylitol, glucitol, ducitol, inositol, arabinitol, arabitol, galactitol, iditol, allitol, maltitol, fructose, sorbose, glucose, xylose, trehalose, allose, dextrose, altrose, lactose, glucose, fructose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, sucrose, maltose, lactose, lactulose, fucose, rhamnose, melibiose, maltotriose, raffinose, allitol, their optically active forms (D- or L-forms) and the corresponding racemates; inorganic salts, both minerals and organominerals such as calcium salts such as lactate, gluconate, glycerolphosphate, citrate, monobasic and dibasic phosphates, succinate, sulfate and tartrate, and the same salts of aluminum and magnesium; carbohydrates such as conventional monosaccharides and disaccharides and the corresponding polyhydric 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, dextrose, lactose, sucrose, polyvinylpyrrolidone, trehalose, glucose and combinations thereof. A particularly useful bulking agent is dextran.

[0035] The term "pharmaceutically acceptable salt", as used herein, is recognized in the art and refers to relatively non-toxic inorganic and organic acid addition salts or inorganic or organic base addition salts of a compound, including those 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 materials used in pharmaceutical formulations.

[0036] The term "substitution", as used herein, refers to the replacement of an amino acid in a sequence by a different amino acid. As used herein, the abbreviation X10Y indicates that the amino acid X found at position 10 of the sequence has been replaced by the amino acid Y. As an example, W26C indicates that the amino acid tryptophan-26 (Trp, W) has been changed to cysteine (Cys). Similarly, the notation AA X indicates that AA is the amino acid that replaces the amino acid found at position X. As an example, Lys 26 indicates the substitution of the amino acid at position 26 of the sequence with lysine. The use of any abbreviation is interchangeable. Further, the use of one-letter or three-letter abbreviations for amino acids is also interchangeable.

[0037] As used herein, the term "therapeutic agent" refers to any chemical or biochemical moiety that is a biologically, physiologically or pharmacologically active substance recognized in the art and that acts locally or systemically in a subject. Examples of therapeutic agents, also referred to as "drugs", are described in well-known literature such as the Merck Index, the Physician’s Desk Reference and The Pharmacological Basis of Therapeutics, and include, without limitation, agents; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, treatment or alleviation of a disease or disorder; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a physiological environment.

[0038] As used herein, the term "therapeutic effect" refers to a local or systemic effect in an animal, particularly a mammal and especially a human, caused by a pharmacologically active substance. Thus, the term means any substance intended for use in the diagnosis, treatment, alleviation, management or prevention of a disease or disorder, or in the promotion of a desired physical or mental manifestation and / or condition in an animal or human. The phrase "therapeutically effective amount" means an amount of such a 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 a substance varies depending on the subject and disease state being treated, the body weight and age of the subject, the severity of the disease state, the route of administration, etc., and can be readily determined by one of ordinary skill in the art. For example, a composition of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0039] As used herein, the term "treating" includes alleviating, reducing, modulating, or eliminating any effect, e.g., amelioration of a condition, disease, disorder, etc. As used herein, "treating" can include both prophylactic and therapeutic treatment. For example, therapeutic treatment can include delaying, inhibiting, or preventing the progression of cystic fibrosis or non-small cell lung cancer, and alleviating or eliminating symptoms associated with cystic fibrosis or non-small cell lung cancer. Prophylactic treatment can include preventing, suppressing, or delaying the onset of cystic fibrosis or non-small cell lung cancer.

[0040] As used herein, "effective amount" refers to an amount sufficient to induce 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).

[0041] "Buffer", as used herein, is any combination of an acid or salt that is pharmaceutically acceptable and that can maintain the composition of the present invention within a desired pH range. The buffer in the disclosed composition maintains the pH in the range of 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 that can maintain the above pH ranges, such as, for example, acetic acid, tartaric acid, phosphoric acid, or citric acid buffer. In one embodiment, the buffer is a phosphate buffer. In another embodiment, the buffer is an acetate buffer. In one embodiment, the buffer is dibasic sodium phosphate, sodium chloride, potassium chloride, and monobasic potassium phosphate.

[0042] In the disclosed composition, the concentration of the buffer generally ranges 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.

[0043] As used herein, "antimicrobial agent" is a pharmaceutically acceptable preservative suitable for administration to a subject that inhibits, prevents, or delays the growth or microorganisms, including for example bacteria, viruses, and fungi, in the compositions of the present invention. Suitable antimicrobial agents for use in the compositions and methods of the present invention include, but are not limited to, cresol, benzyl alcohol, phenol, benzalkonium chloride, benzethonium chloride, chlorobutanol, phenylethyl alcohol, methylparaben, propylparaben, thimerosal, and phenylmercuric nitrate and 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.

[0044] As used herein, an effective amount of an antimicrobial agent is an amount effective to inhibit, prevent, or delay the growth or microorganisms, including for example bacteria, viruses, and fungi, in the compositions of the present invention. In the compositions of the present invention, the amount of the antimicrobial agent is generally in the range of 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.

[0045] The compositions of the present invention can also be lyophilized using lyophilization techniques known in the art and stored as a powder that can be reconstituted prior to administration. The term "lyophilization" as used herein includes a lyophilization or dehydration technique that removes the solvent, preferably a water-miscible solvent, more preferably water, from the composition or the present invention, generally by sublimation under high vacuum when the composition is in a frozen state. Generally, lyophilization is carried out in a lyophilization apparatus (lyophilizer), which includes a drying chamber with variable temperature control, a condenser for recovering water, and a vacuum system for reducing the pressure in the drying chamber.

[0046] The term "lyophilized composition", as used herein, means a solid residue or powder that is produced or remains after the lyophilization procedure as described above. The lyophilized compositions of the present invention generally further comprise pharmaceutically acceptable excipients. The term "pharmaceutically acceptable excipient", as used herein, refers to a substance that is added to the solution prior to lyophilization to enhance characteristics of the lyophilized cake such as color, texture, strength and volume. Pharmaceutically acceptable excipients can be, for example, buffers and pH adjusters, crystalline bulking excipients, stabilizers and tonicity raising agents.

[0047] 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 glycerin or propylene glycol or mixtures thereof and albumin.

[0048] A pharmaceutically acceptable salt is a salt suitable for administration to a subject, such as a human. The chimeric nuclease of the present invention may have one or more sufficiently acidic protons that can react with a suitable organic or inorganic base to form a base addition salt. Examples of base addition salts include those derived from inorganic bases such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc. and organic bases such as alkoxides, alkylamides, alkyl and aryl amines. Thus, such bases useful in preparing the salts of the present invention may include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, etc. The chimeric nuclease of the present invention having a sufficiently basic group such as an amine can react with an organic or inorganic acid to form an acid addition salt. Acids commonly used to form acid addition salts from compounds having a basic group 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-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of such salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, etc.

[0049] Although specific embodiments of the present invention have been discussed, the above specification is illustrative and not restrictive. Upon reviewing this specification, many variations of the present invention will become apparent to those skilled in the art. The full scope of the present invention should be determined by reference to the claims, along with such variations, equivalents, and the entire scope of the specification.

[0050] Unless otherwise indicated, all numbers expressing amounts of ingredients, reaction conditions, and the like, set forth in this specification and claims, are to be understood as being 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.

[0051] The above considerations are to be regarded as illustrative of the principles of the present invention and examples of various embodiments. Once the above disclosure is fully understood, variations and modifications of the numbers will be apparent to those skilled in the art. The following claims are intended to be construed as encompassing all such variations and modifications.

[0052] Abbreviations The abbreviations used in this specification are defined as follows: AA Amino acid Cas9 CRISPR-associated protein 9 CF Cystic fibrosis CFTR Cystic fibrosis transmembrane conductance regulator gene cjCas9 Campylobacter jejuni Cas9 Cpf1 CRISPR from Prevotella and Francisella 1 CRISPR Clustered regularly interspaced short palindromic repeats DLS Dynamic light scattering DMEM Dulbecco's modified Eagle's medium DMPE 1,2-Ditetradecanoyl-sn-glycero-3-phosphoethanolamine DNA Deoxyribonucleic acid DOAB Dioctadecyldimethylammonium bromide DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine DPPC Dipalmitoylphosphatidylcholine E.coli Escherichia coli EDTA Ethylenediaminetetraacetic acid EGFR Epidermal growth factor receptor ELISA Enzyme-linked immunosorbent assay fnCas9 Francisella novicida Cas9 HDR Homology-directed repair IMAC Immobilized metal affinity chromatography IPTG Isopropyl β-D-1-thiogalactopyranoside MPEG-5000-DMPE N-(Carbonyl-methoxypolyethylene glycol 5000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine NSCLC Non-small cell lung cancer NHEJ Non-homologous end joining NLS Nuclear localization signal PC Phosphatidylcholine PCR Polymerase chain reaction PE Phosphoethanolamine RNA Ribonucleic acid saCas9 Staphylococcus aureus Cas9 scCas9 Streptococcus canis Cas9 SDS Sodium dodecyl sulfate spCas9 Streptococcus pyogenes Cas9 TALEN Transcription activator-like effector nuclease TEV Tobacco etch virus TevCas9 Modified I-TevI domain, linker peptide, and modified RNA-guided nuclease Staphylococcus aureus Cas9 ZFN Zinc-finger nuclease

[0053] The inventors discovered a chimeric nuclease comprising a modified version of the I-TevI domain, a linker peptide, and a modified version of the RNA-guided nuclease Staphylococcus aureus Cas9 (hereinafter referred to as "TevCas9" herein), which, when delivered to cells when mixed with lipid nanoparticles with or without exogenous donor DNA, replaces a DNA sequence in the presence of exogenous donor DNA or deletes a DNA of a defined length in the absence of exogenous donor DNA. The novel chimeric nuclease has been shown to edit genes in either whole organisms (in vivo) or isolated cell cultures (ex vivo), in cells of other organisms such as bacteria, yeast, insects, plants, or other mammals, as well as in human cells.

[0054] The novel chimeric nuclease discovered by the inventors outperforms existing gene editing techniques and methods and, in particular, exhibits the following advantages. a. The nuclease, which is a modified version of the TevCas9 nuclease, can target two independent target sites as a single protein and can cleave DNA at one or both of these sites. This can be reprogrammed to many different target DNA sequences by modifying one or more of the I-TevI domain, the linker domain, the Cas9 domain, or the guide RNA (which targets the Cas9 domain to its target sequence). b. When the nuclease cleaves at two sites, this excises a DNA of an exact length (~30 - 36 bases depending on the sites targeted by I-TevI and Cas9); c. The Cas9 domain contains a mutation (D10E) that is rationally designed to modify Cas9 nuclease activity and / or improve the specificity of the Cas9 domain for its target binding site. d. In the presence of exogenous donor DNA, the present invention is designed to replace the target DNA sequence at a higher percentage of cells than existing techniques or practices. e. The nuclease can be purified as a single proximity protein combined with guide RNA, which simplifies production; f. Lipid nanoparticles enable non-viral delivery to target cells with high efficiency and low toxicity, allowing for controlled administration of the nuclease. While other lipid-based nuclease delivery technologies exist, there is no composition suitable for in vivo use; g. Lipid nanoparticles are also designed for delivery of the nuclease through inhalation; h. One version of the nuclease targets and cleaves the CFTR gene to correct the CFTR delta F508 mutation (SEQ ID NO: 1) for the treatment of cystic fibrosis; i. Another version of the nuclease is designed to target and cleave clinically relevant EGFR exon 19 deletion mutations (SEQ ID NOs: 2 - 4) present in various cancers, including non-small cell lung cancer (NSCLC).

[0055] Fusions of GIY-YIG nucleases, such as I-TevI, through a flexible linker to a DNA binding domain are known (WO 2014 / 121222). A previous version of the dual-cutting TevCas9 has been described, which includes amino acids 1-92 of the wild-type I-TevI nuclease domain, a linker region including 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 include: i. An I-TevI nuclease domain that binds to a new target sequence that enables targeting clinically relevant gene sequences, such as the CFTR gene; ii. Various flexible linker regions that are intended to confer different DNA binding or nuclease activities to TevCas9; iii. The saCas9 nuclease domain (US Patent Application No. 1988 / 065406B2). The use of saCas9 over spCas9 results in a smaller DNA coding sequence (~3.7 kilobases for Tev-saCas9 versus ~4.6 kilobases for Tev-spCas9) and a lower molecular weight TevCas9 protein (~144 kilodaltons for Tev-saCas9 versus ~179 kilodaltons for Tev-spCas9), which is suitable for multiple delivery techniques; cleavage by the saCas9 domain between the third and fourth nucleotides is predictable as discovered by the inventors of the claimed technology, which is suitable for deletions of defined length. iv. One version where the guide RNA is targeted to a specific CFTR gene sequence near the CFTR deltaF508 mutation; and v. A second version where the guide RNA is targeted to a specific EGFR gene sequence and is intended to cleave only DNA having an appropriated separated I-TevI site and Cas9 target site. Such appropriately separated sites are present in certain EGFR exon 19 deletion mutations (SEQ ID NOs: 2-4) but not in wild-type EGFR (SEQ ID NO: 5); 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 can deliver nucleases to cells with high efficiency and low toxicity; b. A pharmaceutical formulation of lipid, nuclease, and exogenous donor DNA; c. A pharmaceutical formulation of lipid, nuclease, and exogenous donor DNA that is suitable for spraying (inhalation); and d. A version of the present invention containing exogenous donor DNA that is integratable between or in the regions surrounding two sites targeted by the nuclease when delivered together with the TevCas9 nuclease in lipid nanoparticles.

[0056] The novel chimeric nuclease composition of the present application contains different combinations of I-TevI domain, linker domain, Cas9 domain, and guide RNA.

[0057] The version targeting the CFTR gene is composed of the following: i. The I-TevI domain of the amino acid sequence according to SEQ ID NO: 6; ii. A linker domain according to any one of SEQ ID NOs: 7-12; iii. The saCas9 domain of the amino acid sequence according to SEQ ID NO: 13; and iv. The guide RNA of the RNA sequence according to SEQ ID NO: 15 or 21.

[0058] The version targeting the EGFR gene is composed of the following: i. The I-TevI domain of the amino acid sequence according to SEQ ID NO: 6; ii. A linker domain having any one of the amino acid sequences according to SEQ ID NOs: 7 to 12; iii. The saCas9 domain of the amino acid sequence according to SEQ ID NO: 13; and iv. The guide RNA of the RNA sequence in SEQ ID NO: 16.

[0059] The I-TevI domain of the preferred embodiment is the 93-amino acid I-TevI domain of Escherichia coli phage T4 according to the following sequence: MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIA (SEQ ID NO: 6)

[0060] The saCas9 of the preferred embodiment is a polypeptide composed of 1,053 amino acids according to the following sequence:

[0061] Glu of the preferred embodiment 10 The saCas9 having the mutation is a polypeptide composed of 1,053 amino acids according to the following sequence (the mutation is underlined): MKRNYILGL E

[0062] The version of guide RNA targeting the CFTR gene is composed of 101 ribonucleotides with the following sequence: GCGUCAUCAAAGCAUGCCAACGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 15) AUAUCAUUGGUGUUUCCUAUGGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 21)

[0063] The version of guide RNA targeting the EGFR gene is 101 ribonucleotides in length with the following sequence: AAUUUUAACUUUCUCACCUUCGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU (SEQ ID NO: 16).

[0064] The linker used in any of the above constructs can be selected from the group consisting of:

Table 1

[0065] Synthetic Example 1: Method for producing TevCas9 nuclease: The DNA coding sequences of the above I-TevI domain, linker domain, and Cas9 domain are synthesized as one contiguous DNA sequence using techniques known in the art. Gene synthesis was performed by Bio Basic Inc. (Markham, On, Canada). Briefly, short oligonucleotides (~50 - 60 base pairs) containing overlapping regions to cover the entire sequences of the I-TevI domain, linker domain, and Cas9 domain are synthesized. The oligonucleotides are mixed together in approximately 1 kilobase blocks of the sequence to be synthesized, and these ~1 kilobase blocks are synthesized using polymerase chain reaction (PCR). These ~1 kilobase blocks are then mixed and subjected to PCR to synthesize the I-TevI domain, linker domain, and Cas9 domain. Further, the DNA sequence of TevCas9 was optimized prior to synthesis to facilitate expression of TevCas9 in Escherichia coli (E. coli) and to simplify restriction enzyme digestion. First, three-base pair DNA codons that are not used very frequently by Escherichia coli (E. coli) were replaced with those that occur more frequently (for example, for the six codons encoding the amino acid arginine, the relative abundance of the codon AGG is 0.03 compared to 0.42 for the codon CGT). In total, 37% of the codons were changed to those preferred by E. coli. 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, an NdeI restriction endonuclease site was removed from the internal sequence. The contiguous DNA is digested with the restriction endonucleases NdeI and BamHI (New England Biolabs, Ipswich, MA, United States), where the target site appears only once in its DNA sequence, and then inserted into the similarly digested pET-11a expression vector (EMD Millipore, Burlington, MA, United States) suitable for expression of TevCas9 in E. coli using DNA ligase (New England Biolabs, Ipswich, MA, United States).The pET-11a vector containing TevCas9 is transformed into the E. coli expression strain T7 Express (New England Biolabs #C2566, Ipswich, MA, United States), which is optimized for protein expression and contains a nuclease. Alternatively, the E. coli expression strain BL-21(DE3) (New England Biolabs #C2527, Ipswich, MA, United States) is used. The success of the transformation is confirmed by the resistance of E. coli to ampicillin or tetracycline, and the coding sequence of TevCas9 is verified by DNA sequencing of the expression vector derived from the transformed E. coli. The transformed E. coli is grown at 37 °C to an optical density of 0.4 - 0.6 measured by a spectrophotometer at a wavelength of 600 nM, and the expression of TevCas9 protein from the pET-11a vector in the transformed E. coli expression strain is induced using IPTG at 16 °C for 10 - 12 hours. The successful expression of TevCas9 is verified by the presence of a band of approximately 150 kDa on a Coomassie-stained SDS-polyacrylamide gel in the sample of the inducer compared to the non-induced sample. The E. coli cells are harvested by centrifugation and resuspended in a lysis buffer containing 10 mM imidazole (Sigma, St. Louis, MO, United States), 300 - 500 mM sodium chloride (Sigma-Aldrich, St. Louis, MO, United States) and 50 mM disodium phosphate (Sigma, St. Louis, MO, United States), pH 8.0 [Buffer 1]. Alternatively, the disodium phosphate in Buffer 1 is replaced with 10 mM Tris-HCl (Sigma, St. Louis, MO, United States), pH 8.E. coli is lysed by homogenization using any other suitable lysis method known in the art, such as high-pressure liquid operated at 600 - 1000 bar, a homogenizer (Avestin Inc., Ottawa, ON, Canada) or sonication using a sonicator (Branson Ultrasonics Corp, Danbury, CT, United States) with lysozyme treatment, homogenization using a French pressure cell (Glen Mills Inc., Clifton, NJ, United States) or homogenization using a Dounce homogenizer (Corning Inc., Corning, NY, United States). The lysed material is centrifuged at 12,000 rpm for 20 - 30 minutes at 4°C, and the supernatant containing soluble TevCas9 is used in the subsequent purification steps. The pellet contains cell debris, insoluble intracellular material and any insoluble TevCas9. The success of lysis and solubility are verified by the presence of a band of approximately 150 kDa on a Coomassie-stained SDS-polyacrylamide gel in the supernatant sample when compared to a resuspended sample of the pellet.

[0066] The TevCas9 nuclease is purified in the following steps: 1. The lysate containing the nuclease is loaded onto an immobilized metal affinity chromatography (IMAC) column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) that binds the nuclease. 2. The IMAC column is washed with Buffer 1. 3. Elute the TevCas9 still bound to the column with a solution containing 250 mM imidazole (Sigma, St. Louis, MO, United States), 300 mM - 500 mM sodium chloride (Sigma - Aldrich, St. Louis, MO, United States) and 50 mM sodium phosphate (dibasic) (Sigma, St. Louis, MO, United States), pH 7.6 - 8.0 [Buffer 2]. Alternatively, replace the sodium phosphate (dibasic) in Buffer 2 with 10 mM Tris - HCl (Sigma, St. Louis MO, United States), pH 7.6 - 8. 4. Treat the eluate with tobacco etch virus (TEV) protease (New England Biolabs, Ipswich, MA, United States) and incubate with the appropriate guide RNA. The guide RNA is synthesized by Integrated DNA Technology Inc. (Coralville, IA, United States). 5. Load the treated eluate onto the IMAC column again and collect the flow - through containing TevCas9 nuclease and guide RNA. 6. Confirm the success of TevCas9 nuclease purification by the presence of a 150 - kilodalton protein band on a Coomassie - stained SDS - polyacrylamide gel. Confirm the success of co - purification of TevCas9 with the guide RNA by treating a sample of the eluate with proteinase K (New England Biolabs, Ipswich, MA, United States), then split the sample into two, treat one sub - sample further with RNase A (New England Biolabs, Ipswich, MA, United States), and keep the other in a control buffer without RNase A. A ~ 100 - nucleotide RNA band is visible on a urea - polyacrylamide gel in the control sample but not in the RNase A - treated sample. 7. Dialyze the solution containing TevCas9 nuclease and guide RNA against a solution containing phosphate - buffered saline, pH 7.4.

[0067] Example 2: Method for Producing Lipid Nanoparticles The lipid nanoparticles of the preferred embodiment are composed of one of the following mixtures: I. Lipid nanoparticle No. 1 contains DOPE (Avanti Polar Lipids, Alabaster, AL, United States) and MPEG-5000-DMPE (Avanti Polar Lipid, Alabaster, AL, United States) in a molar ratio of 2:0.05, respectively; II. Lipid nanoparticle No. 2 contains DPPC (Avanti Polar Lipid, Alabaster, AL, United States), cholesterol (SUPELCO, Bellefonte, PA, United States) and DOBA (Sigma, St. Louis, MO, United States) in a molar ratio of 7:2:1, respectively; and III. Lipid nanoparticle No. 3 contains DPPC, cholesterol and MPEG-5000-DMPE (Avanti Polar Lipid, Alabaster, AL, United States) in a molar ratio of 4:1:0.125, respectively.

[0068] The lipid nanoparticles are produced to have an average diameter of approximately 100 nM.

[0069] Select one of lipid mixtures numbered 1 to 3. For example, DOPE and MPEG-5000-DMPE are mixed together in an appropriate molar ratio in an organic solvent such as chloroform. The organic solvent is then evaporated and the dried lipid mixture is resuspended by vigorously vortexing in a solution containing phosphate buffered saline, pH 7.4. The resuspended lipid mixture is then 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 by passing it 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 Ltd, Malvern, United Kingdom) or by another suitable technique known in the art.

[0070] Example 3: Composition of Donor DNA Donor DNA contains a DNA sequence intended to repair a genetic abnormality. This also includes DNA sequences not found in the target genomic DNA; these sequences do not interfere with normal gene function but are intended to knock out the I-TevI and / or Cas9 sites and / or introduce one or more DNA sequences used to track the success of target gene repair. Examples of donor DNA include, but are not limited to: Linear single-stranded DNA of various lengths containing a homologous region adjacent to the site targeted / cut by I.TevCas9 Linear double-stranded DNA of various lengths containing a homologous region adjacent to the site targeted / cut by II.TevCas9; Double-stranded DNA of the same length that is cut by a nuclease and also contains a DNA end complementary to that cut by TevCas9; Circular double-stranded DNA containing a homologous region adjacent to the site targeted / cut by TevCas9; and A circular double-stranded DNA containing an I-TevI target site and a Cas9 target site, wherein the product cleaved from the V.2 native strand DNA contains ends complementary to those cleaved by TevCas9.

[0071] Example 4: Method for assembling lipid-encapsulated TevCas9 and transfecting cells For ex vivo cell transfection: To assemble lipid-encapsulated TevCas9, lipid nanoparticles are mixed with TevCas9 at a molar ratio of 2000:1 in Dulbecco's Modified Eagle Medium (DMEM) (Sigma, St. Louis, MO, United States) and incubated at room temperature for 10 minutes. Cells are transfected using 8.7x10E-17 to 3.1x10E-17 moles of lipid-encapsulated TevCas9 / cell.

[0072] For in vivo cell transfection: To assemble lipid-encapsulated TevCas9, lipid nanoparticles are 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 / cell for in vivo transfection is determined.

[0073] Other embodiments The nuclease can contain different combinations of the I-TevI domain, linker domain, Cas9 domain, or guide RNA, as highlighted below.

[0074] Modification of the I-TevI domain: Other versions of the I-TevI nuclease domain can contain different combinations of mutations that change the sequence recognized by I-TevI, such as K26 and / or C39, mutations that change the site targeted by the I-TevI domain, or mutations that change the activity of the I-TevI domain. Other versions of the nuclease can replace the I-TevI domain with other GIY-YIG nuclease domains, such as I-BmoI, Eco29kI. Other versions do not contain Met as a result of processing when expressed in E. coli. 1 does not contain.

[0075] Modification of the linker domain: The linker domain includes the following and may further include one of the following that changes the binding specificity or activity of TevCas9: a) an I-TevI linker domain containing one or more mutations to 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.

[0076] Modification of the Cas9 domain: Other versions of the Cas9 domain may contain the following: a) a version of the saCas9 domain containing the D10E mutation (SEQ ID NO: 14); b) a version of the saCas9 domain that makes a cut in 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 to the target DNA but does not cleave it, such as a mutation with both the D10A and H557A mutations (SEQ ID NO: 18); d) a version of the previously described spCas9EQR variant containing the mutations D1135E, R1335Q and T1337R combined with the D10E mutation (SEQ ID NO: 19); and e) a version of the previously described spCas9EQR variant containing the mutations D1135E, R1335Q and T1337R combined with the D10E mutation and a mutation that makes a cut in 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 and do not contain.

[0077] Other versions may replace the Cas9 domain with other nucleases or DNA binding domains, such as: a) meganucleases, such as the family LAGLIDADG, 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 the zinc-finger motif, TALE activator domain, etc.

[0078] Modification of guide RNA: a) Other versions of the guide RNA may target the same region of DNA in the CFTR gene or the EGFR gene but contain different sequences that constitute genetic polymorphisms in the population; b) Other versions of the guide RNA may target different sequences in the CFTR gene or the EGFR gene; c) Other versions of the guide RNA may target other sequences in the genome to retarget the nuclease to additional clinically relevant targets; d) Other versions of the guide RNA may contain bridged nucleic acids (“BNA”) that promote target site specificity; and e) Other versions may contain a mixture of guide RNAs that target multiple sequences within the same gene.

[0079] Modification of lipid nanoparticles: a) Other versions of lipid nanoparticles number 1, 2, or 3 may have different ratios of each lipid component; b) Other versions of lipid nanoparticles may have different average diameters; c) Other versions of lipid nanoparticles may contain different cationic or neutral lipids; d) Other versions of lipid nanoparticles may contain peptides that target specific cell types; e) Other versions of lipid nanoparticles may contain compounds that bind DNA, such as GL67(N 4 -cholesteryl-spemine); f) Lipid nanoparticles may be lyophilized for stability promotion; and g) Lipid nanoparticles may be resuspended in solutions other than phosphate-buffered saline, such as sterile isotonic saline, water for injection, etc.

[0080] Modification of the composition of donor DNA: a) Other versions of linear double-stranded donor DNA may contain longer regions of single-stranded DNA that are complementary to the target sequence; and b) Other versions of circular double-stranded DNA may contain other DNA sequences that are intended to increase the ratio of homologous recombination modification.

[0081] Variant methods for assembling and transfecting cells with lipid-encapsulated nucleases: 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 10 minutes or for more than 10 minutes; and d) Other molar amounts of nuclease / cell may be used in the transfection reaction.

[0082] Alternative methods for producing nucleases: a) other E. coli expression strains such as LS5218 (Escherichia coli Genetic Stock Center - Yale University, New Haven, CT, United States) or BL21-DE3 (New England Biolabs, Ipswich, MA, United States) can be used; b) Buffer 1 or 2 can contain different concentrations of imidazole, sodium chloride, sodium phosphate (dibasic) or tris hydrochloride and be buffered to different pH values; c) Other processing steps such as cation or anion exchange chromatography can be used; d) The nuclease can be dialyzed into solutions other than phosphate buffered saline, such as sterile isotonic saline, water for injection, etc.; e) The nuclease can be lyophilized for stability promotion; f) Guide RNA can be co-expressed from the pACYC-Duet1 expression vector (EMD Millipore, Burlington, MA, United States). The DNA coding sequence of the guide RNA is synthesized (Integrated DNA Technology Inc., Coralville, IA, United States), digested with restriction endonucleases and inserted into a second expression site similarly digested in the pACYC-Duet1 expression vector; g) Guide RNA can be synthesized from double-stranded DNA by transcribing the guide using the T7 RNA Polymerase HiScribe Kit (New England Biolabs #E2040S, Ipswich, MA, United States) and purifying the guide using the RNA Cleanup Kit (New England Biolabs #T2030L, Ipswich, MA, United States).

[0083] Test Example 1: Method for demonstrating correction of the function of CFTR delta F508 and CFTR protein in a model cell line Cultures of immortalized epithelial cells homozygous for the CFTR deltaF508 mutation, such as the CuFi-1 cell line (ATCC® CRL-4013™, American Type Culture Collection, Manassas, VA, United States), are treated with various concentrations of lipid-encapsulated TevCas9 and donor DNA (Specific Biologics, Toronto, ON, Canada) in a pharmaceutical formulation targeted to the CFTR deltaF508 mutation. Appropriate control cell lines, such as the NuLi-1 (ATCC® CRL-4011™, American Type Culture Collection, Manassas, VA, United States) immortalized epithelial cells homozygous for wild-type CFTR, are also used.

[0084] The percentage of cells with corrected CFTR deltaF508 relative to uncorrected cells is measured by the T7 endonuclease I assay (EnGen® Mutation Detection Kit, New England Biolabs #E3321, Ipswich, MA, United States), restriction endonuclease digestion of the PCR-amplified target site (New England Biolabs, Ipswich, MA, United States), deep gene sequencing using the Illumina MiSeq system and barcoded primers adjacent to the target site (Illumina, San Diego, CA, United States), or other suitable methods. The effect of TevCas9 treatment in a control cell line (e.g., NuLi-1 (ATCC® CRL-4011™, American Type Culture Collection, Manassas, VA, United States)) is evaluated. CFTR functionality is measured in treated CuFi-1 cultures (ATCC® CRL-4013™, American Type Culture Collection, Manassas, VA, United States) versus mock-treated CuFi-1 cultures (ATCC® CRL-4013™, American Type Culture Collection, Manassas, VA, United States) using short-circuit current measurements in an Ussing Chamber (Warner Instruments, Hamden, CT, United States) in the presence of a chloride ion gradient. The effect of TevCas9 treatment in a control cell line (e.g., NuLi-1) is also measured.

[0085] To demonstrate EGFR exon 19 deletion mutations and disruption of EGFR expression and activity in model cell lines, cultures of immortalized epithelial cells expressing EGFR exon 19 deletion mutations, such as the HCC827 cell line (ATCC® CRL-2868™, American Type Culture Collection, Manassas, VA, United States), are treated in the concentration range of lipid-encapsulated TevCas9 (Specific Biologics, Toronto, ON, Canada) in a pharmaceutical formulation of phosphate-buffered saline, sterile isotonic saline, or water for injection that is targeted to the EGFR exon 19 deletion. Appropriate control cell lines, such as NuLi-1 (ATCC® CRL-4011™, American Type Culture Collection, Manassas, VA) or an immortalized epithelial cell line that is homozygous for wild-type EGFR, are used.

[0086] The percentage of cells with disrupted EGFR exon 19 deletions relative to non-corrected cells is measured by the T7 endonuclease I assay (EnGen® Mutation Detection Kit, New England Biolabs #E3321, Ipswich, MA, United States), restriction endonuclease digestion of the PCR amplified target site (New England Biolabs, Ipswich, MA, United States), deep gene sequencing using the Illumina MiSeq system and barcoded primers adjacent to the target site (Illumina, San Diego, CA, United States) or other suitable methods. The EGFR protein expression and activity, and the effect of TevCas9 treatment in a control cell line (e.g., NuLi-1 (ATCC® CRL-4011™, American Type Culture Collection, Manassas, VA, United States)) are also evaluated. Phosphorylated (i.e., activated), non-phosphorylated, and total EGFR proteins are detected in treated HCC827 cultures (ATCC® CRL-2868™, American Type Culture Collection, Manassas, VA, United States) versus mock-treated HCC827 cultures using an enzyme-linked immunosorbent assay (ELISA) (Sigma, St. Louis, MO, United States). The effect of TevCas9 treatment in a control cell line (e.g., NuLi-1 (ATCC® CRL-4011™, American Type Culture Collection, Manassas, VA, United States)) is also evaluated.

[0087] Example 1: Animal model studies planned to demonstrate efficacy and determine dose-limiting toxicity In an exemplary method for demonstrating the correction of CFTR deltaF508 and / or symptoms of cystic fibrosis using lipid-encapsulated TevCas9 treatment in an animal model (e.g., mouse, rat, mini-pig or ferret), lipid-encapsulated TevCas9 in a pharmaceutical formulation of phosphate buffered saline, sterile isotonic saline or water for injection, targeted to CFTR deltaF508, is delivered directly to the lungs by either intubation or intranasal delivery. The procedure time is approximately 30 - 6000 seconds / treatment, depending on the animal model used.

[0088] Alternatively, lipid-encapsulated TevCas9 in a pharmaceutical formulation targeted to CFTR delta F508 is sprayed using a commercially available nebulizer (Aeroneb®, AeroEclipse®, (Trudell Medical, London, ON, Canada)) or PARI-LC Plus®, (PARI USA, Midlothian, VA, United States)). The average particle size of the lipid nanoparticles, approximately 100 nM, is confirmed after spraying by dynamic light scattering (DLS) using a Zetasizer (Malvern Panalytical Ltd, Malvern, United Kingdom) or another suitable technique known in the art. The composition and concentration of the lipid-encapsulated TevCas9 are confirmed after spraying using a MicroGram Lipid Assay Kit (ProFoldin, Hudson, MA, United States) and the presence of a band at approximately 150 kDa on a Coomassie-stained SDS-polyacrylamide gel. For measurement of the gene correction ratio, a representative ovine (miniature pig) animal model (Exemplar Genetics, Sioux City, IA, United States) that is homozygous for the CFTR delta F508 mutation is exposed orally, nasally, or directly into the lungs to lipid-encapsulated TevCas9 targeted to CFTR delta F508 and appropriate controls. General maintenance of these animals includes breeding and parturition; age-appropriate bio-secure containment; sound nutrition; basic vaccination and veterinary care; and documentation in accordance with animal welfare guidelines. Maintenance of these animals specific to CFTR delta F508 may include one or more of the following: surgery to address intestinal obstruction; pancreatic enzyme replacement therapy; vitamins and H2 blockers; and / or proton pump inhibitors to improve gastric acid regulation. Miniature pigs are treated for 2 days to 4 weeks for acute toxicity testing and up to 24 months for chronic toxicity testing in the concentration range of lipid-encapsulated TevCas9 predicted to be effective from the above model cell line experiments.

[0089] Animals are monitored after treatment to evaluate the overall health status of the animals for any treatment-related adverse events such as changes in behavior, body weight or diet consumption; immune response; changes in the health status of the cardiovascular system; mortality, etc. Other measures of effectiveness after treatment may include: I. Forced respiration in each animal after treatment, such as forced expiratory volume (or other appropriate method); II. Overall survival of each animal compared to the control; III. Other measures of lung function (e.g., use of a mechanical ventilator that can perform a general lung function assessment); and IV. Measurement of mutations in vivo through tissue sampling and mutation detection methods such as by polymerase chain reaction.

[0090] After treatment with lipid-encapsulated TevCas9, the animals are sacrificed and the lung and trachea tissues are recovered.

[0091] The proportion of cells with corrected CFTR delta F508 relative to non-corrected cells is measured by the T7 endonuclease I assay (EnGen® Mutation Detection Kit, New England Biolabs #E3321, Ipswich, MA, United States), restriction endonuclease digestion of the PCR-amplified target site (New England Biolabs, Ipswich, MA, United States), deep gene sequencing using the Illumina MiSeq system and barcoded primers adjacent to the target site (Illumina, San Diego, CA, United States), or other appropriate methods.

[0092] In a method for demonstrating disruption of the EGFR exon 19 deletion mutation and / or non-small cell lung cancer (NSCLC) symptoms by TevCas9 treatment in an animal model, lipid-encapsulated TevCas9 targeted to the EGFR exon 19 deletion mutation in a pharmaceutical formulation of phosphate buffered saline, sterile isotonic saline, or water for injection is delivered directly to the lung through the oral cavity, nose, or directly to the lung. The procedure time is approximately 30 - 6000 seconds / treatment depending on the animal model used.

[0093] In another method, lipid-encapsulated TevCas9 targeted to the EGFR exon 19 deletion mutation in a pharmaceutical formulation is sprayed using a commercially available nebulizer ((Aeroneb®, AeroEclipse®, (Trudell Medical, London, ON, Canada) or PARI-LC Plus®, (PARI USA, Midlothian, VA, United States)). The average particle size of the lipid nanoparticles of approximately 100 nM is confirmed after spraying by dynamic light scattering (DLS) using a Zetasizer (Malvern Panalytical Ltd, Malvern, United Kingdom) or other suitable techniques known in the art. The composition and concentration of the lipid-encapsulated TevCas9 nanoparticles are confirmed after spraying using a MicroGram Lipid Assay Kit (ProFoldin, Hudson, MA, United States) and the presence of a band of approximately 150 kDa on a Coomassie-stained SDS-polyacrylamide gel. For measurement of the gene disruption ratio, a representative murine (mouse) animal model that is homozygous for the EGFR exon 19 deletion mutation is exposed to lipid-encapsulated TevCas9 targeted to the EGFR exon 19 deletion either through the nose, mouth, or by direct lung exposure. The mice are treated with various concentrations of TevCas9 predicted to be effective from the model cell line experiments for 2 days to 4 weeks for acute toxicity testing and up to 24 months for chronic toxicity testing.

[0094] The overall health status of the animals is monitored after treatment to evaluate any treatment-related adverse events such as changes in behavior, body weight, or diet consumption; immune response; changes in the health status of the cardiovascular system; mortality, etc. Other measures of efficacy after treatment may include the following: I. Quantification of EGFR-activated proteins through positron emission tomography (PET) with an EGFR variant tracer; II. Overall survival of each animal compared to a control; and III. Measurement of tumor formation / regression in each animal over time. Evaluation of mutations in vivo through tissue sample collection and mutation detection methods, such as Cobas® EGFR Mutation Test Version 2 (Roche Diagnostics, Risch-Rotkreuz, Switzerland). After treatment with spray lipid-encapsulated TevCas9, the animals are sacrificed and lung and trachea tissues are harvested.

[0095] The ratio of cells with disrupted EGFR exon 19 deletion mutations to non-disrupted cells is measured by the T7 endonuclease I assay (EnGen® Mutation Detection Kit, New England Biolabs #E3321, Ipswich, MA, United States), restriction endonuclease digestion of the PCR amplified target site (New England Biolabs, Ipswich, MA, United States), deep gene sequencing using the Illumina MiSeq system and barcoded primers adjacent to the target site (Illumina, San Diego, CA, United States), or other appropriate methods. EGFR protein expression and activity in the cells of the harvested tissues are measured using an enzyme-linked immunosorbent assay (ELISA) (Sigma, St. Louis, MO, United States) that detects phosphorylated (i.e., activated), non-phosphorylated, and total EGFR proteins. The determination of the dose-limiting toxicity to enable the first clinical trial in humans is based on the predicted effective dose from the animal model experiments discussed above, and various concentrations of lipid-encapsulated TevCas9 (e.g., milligrams / kilogram body weight) are sprayed and delivered to an appropriate animal model for toxicity testing, such as cynomolgus monkeys or other non-human primates. The overall health status of the animals is monitored for any treatment-related adverse events, such as changes in behavior, body weight, or diet consumption; immune response; changes to the cardiovascular health status; mortality. Other measures of efficacy can be included and measured in this trial.

[0096] Therapeutic effect The novel chimeric nucleases of the present invention are intentionally designed to modify the DNA of lung epithelial cells to treat a monogenic disease, but these can function in vivo or ex vivo, in other cell types or in the cells of other organisms such as bacteria, yeast, insects, plants or other mammals, to treat monogenic or polygenic and infectious diseases.

[0097] Example 1: Method for targeted insertion or substitution of all or part of a DNA sequence in the genome of human cells Figure 2 illustrates the mechanism of action of the novel chimeric nuclease cell uptake of the present invention. As illustrated in Figure 2A, cells 20 (singular or plural) are exposed to novel lipid-encapsulated nuclease particles 21 containing TevCas9 25 by administration either in vivo or ex vivo. As shown in Figure 2B, the lipid-encapsulated nuclease particles 21 are endocytosed into the cells 20. The endosome 22 passes through a maturation process in the cytoplasm and is targeted for degradation (Figure 2C). In certain situations, TevCas9 25 can avoid the endosome 22 and enter the cytoplasm (Figure 2D). In eukaryotes, the nuclease (TevCas9) 25 is targeted to the nucleus 23 of the cell 20 through one or more nuclear localization sequences ("NLSs"). As shown in Figure 2E, through its nuclear localization sequence, TevCas9 25 can enter the nucleus 23, and when in the nucleus 23, the TevCas9 nuclease 25 binds to the target genomic DNA24 sequence and cleaves it 26.

[0098] Figure 3 illustrates the mechanism of TevCas9 nuclease in DNA cleavage. Figure 3A represents important properties of TevCas9 bound to its target genomic DNA sequence 24 as shown before the cleavage reaction. The I-TevI domain 27 targets the I-TevI target sequence 29. The linker domain 30 connects the Cas9 domain 28, which targets the Cas9 target sequence 31, and the I-TevI domain 27. The genetic mutation 32 is surrounded by or in proximity to the I-TevI target sequence 29 and the Cas9 target sequence 31. As shown in Figure 3B, TevCas9 25 cleaves the target sequence, leaving a deletion product 34 of a predictable size along with non-complementary DNA ends 35, 36. Figure 3C illustrates that in the presence of single-stranded donor DNA 37 with homology arms, the cell 20 can insert the donor DNA 37 sequence near the cleavage site through the homologous recombination repair (HDR) pathway 38. Figure 3D illustrates that in the presence of donor DNA 39 with compatible DNA ends for those cleaved by TevCas9 25, the cell 20 can insert the donor DNA sequence 39 between the cleavage sites through directed-ligation using the non-homologous end joining (NHEJ) pathway 40. In the absence of donor DNA, the cell 20 can ligate the DNA ends through the NHEJ pathway 40 (Figure 3E).

[0099] Example 2: Treatment of Cystic Fibrosis In the case of the treatment of cystic fibrosis, the exogenous donor DNA is involved in a method of targeted deletion of a defined length of DNA sequence in human somatic cells to stimulate homologous recombination repair using the exogenous donor DNA as a template, and contains a DNA sequence that repairs the CFTR deltaF508 mutation (Figure 3C).

[0100] Example 3: Treatment of Non-Small Cell Lung Cancer In the case of applications for treating non-small cell lung cancer, a version of the Cas9 domain that cleaves only one strand of DNA (D10A or H557A mutation) or a nuclease deletion version (D10A + H557A mutation) is used, and the target sequence is the EGFR exon 19 deletion mutation (SEQ ID NOs: 2-4). However, in the present application, the nuclease does not contain exogenous donor DNA. In the absence of exogenous donor DNA, cells can remove the DNA sequence between the two sites targeted by the nuclease by non-homologous end joining (Figure 3E).

[0101] Alternatively, the inhalation route is a fast and effective method for the local delivery of drugs to the lungs and for the systemic administration of certain agents. Inhaled drug therapy is widely used to treat respiratory conditions such as asthma and chronic obstructive pulmonary disease (COPD). Research continues to develop inhalation systems for treating cystic fibrosis.

[0102] The following examples are provided to illustrate methods for preparing and using the compounds disclosed herein, but do not limit the scope of the present disclosure in any way. Many other embodiments of this disclosure will be apparent to those skilled in the art.

[0103] An inhaler is a device that delivers drugs to the lungs in the form of aerosolized vapor. Inhalers are commonly used to treat respiratory diseases such as asthma and COPD, such as the inhalation of corticosteroids, but inhalation has also been used to treat and prevent lung infections, such as ARIKAYCE® (Insmed Incorporated, Bridgewater, NJ, United States).

[0104] A nebulizer may require several procedures to prepare the liquid for nebulization. The drug is generally held in liquid form in a cup inside the nebulizer chamber. When switched on, the device generates compressed air to convert the liquid into vapor in the nebulization chamber. The patient places the mouthpiece of the nebulizer chamber in the mouth and takes a sharp, deep inhalation, holding the breath for 5 - 10 seconds to ensure that the drug reaches the lower parts of the lungs. There are various such devices. Many modern nebulizers are breath-activated and rely on the patient's inhalation force to synchronize the aerosolized liquid from the device, thus ensuring that the drug is delivered only to the patient and not to the surrounding environment. This also ensures the consistency of delivery of the full dose of the drug to the patient.

[0105] The use of nebulizers is well-known and nebulizers are commercially available from several sources such as Aeroneb®, AeroEclipse®, (Trudell Medical, London, ON, Canada) or PARI-LC Plus®, (PARI USA, Midlothian, VA, United States). In an embodiment of the present invention, a nebulizer is utilized for the delivery of the novel lipid-encapsulated chimeric nuclease of the present application, comprising 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 the therapeutic agent of interest is placed in the nebulizer chamber, subsequently aerosolized and inhaled into the lungs by the patient via deep breathing.

[0106] Some of the advantages of using a nebulizer over oral or intravenous administration are: the amount of drug required may be less compared to oral or intravenous administration; the onset of action may be more rapid via inhalation compared to the oral route; there may be a lower likelihood of severity of adverse effects due to local delivery of the drug to the lung tissue where the disease manifests itself; inhaled drug therapy is painless and relatively comfortable for the patient, which promotes compliance.

[0107] There is no non-invasive delivery route that provides the rate of action that inhaled drugs can provide. One of the advantages of inhaled drugs is that they are absorbed more rapidly than molecules administered subcutaneously and provide a more immediate physiological response. Small or large molecules, particularly hydrophobic molecules, can be absorbed within seconds of inhalation and can thus be used to treat a wide variety of conditions that occur acutely or require long-term administration. Pain, panic, anxiety, nausea, acute cardiovascular events, bronchoconstriction, sleep induction, convulsions, Parkinson's lock-up and hot flashes are some of the acute conditions that can be addressed with inhaled drugs.

[0108] Most protein-based drug products have some water solubility and are rapidly and effectively absorbed from the lungs. Those with higher hydrophobicity are absorbed even more rapidly within seconds to minutes. Those with higher hydrophilicity are absorbed within minutes to 10 minutes. In one example of the invention, for delivery of a therapeutic dose to the lungs, 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 that is water-soluble and hydrophilic. The doses can range from 1 to 1000 milligrams each of the lipid nanoparticles and the chimeric nuclease, with about 5 to 200 milligrams being preferred. 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 hours, and complete cleavage on a DNA substrate in vitro was observed within 2 hours. Other aerosol therapeutics were administered daily. Aerosol administration of the lipid-encapsulated chimeric nuclease can thus be daily or less frequent, depending on its effectiveness based on the patient. The chimeric nuclease is manufactured by BioVectra Corporation (Charlottetown, PE, Canada) and aseptically filled into vials by Dalton Pharma Services (Mississauga, ON, Canada). The lipid nanoparticles are manufactured by Transferra Nanosciences Inc. (Burnaby, BC, Canada) and aseptically filled into vials.

[0109] The dosage of any of the disclosed compositions will vary depending on 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 subject composition. Any of the subject formulations can be administered in a single dose or in divided doses. The dosage for the present compositions can be readily determined by techniques known to those of skill in the art or as taught herein.

[0110] In certain embodiments, the dosage of the subject compound will generally be in the range of about 1 to 1000 milligrams, specifically in the range of about 5 to 200 milligrams, depending on the weight of the patient.

[0111] Any possible effects on the effective dosage or amount and timing of administration of the present formulation may need to be confirmed for any particular composition of the present disclosure. This can be accomplished by ordinary experimentation as described herein, using one or more groups of animals (preferably at least 5 animals per group), or, if necessary, in human clinical trials. The effectiveness of any subject composition and method for treatment or prevention can be evaluated by administering the composition, measuring one or more applicable indicators, and comparing the post-treatment values of these indicators to the pre-treatment values of the same indicators to assess the effect of the administration.

[0112] The exact administration time and amount of administration of any particular subject composition that will result in the most effective treatment in a patient depends on the activity, pharmacokinetics and bioavailability of the subject composition, the physiological state of the patient (including age, sex, disease type and stage, general health status, response to a particular dosage and type of drug), the route of administration, etc. The guidelines provided herein can be used, for example, to determine the optimal time and / or dosage to optimize treatment, which requires experimentation that does not go beyond ordinary experimentation consisting of monitoring the subject and adjusting the dosage and / or timing.

[0113] While the subject is being treated, the health status of the patient can be monitored by measuring one or more of the relevant indicators at predetermined times during the treatment period. The treatment, including the composition, amount, administration time and prescription, can be optimized according to the results of such monitoring. The patient can be re-evaluated periodically to determine the degree of improvement by measuring the same parameters. Adjustments to the amount of the subject composition administered, and possibly to the administration time, can be made based on these re-evaluations.

[0114] Treatment may be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum therapeutic effect is obtained.

[0115] Use of the subject compositions may reduce the required dosage of any individual agent contained in the composition, since the onset and duration of effect of the different agents may be complimentary.

[0116] The therapeutic efficacy of a subject composition can be determined, for example, by LD 50 and ED 50 This can be determined by standard pharmaceutical procedures in cell cultures or experimental animals to determine

[0117] The data obtained from cell culture assays and animal studies may be used in formulating a range of dosage for use in humans. The dosage of any subject composition is preferably within the range of ED 50 The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. For compositions of the present disclosure, the therapeutically effective dose can be estimated initially from cell culture assays.

[0118] Formulation The pharmaceutical compositions of the present disclosure may be administered by various means depending on their intended use as is well known in the art. For example, the compositions of the present disclosure may be administered via aerosol. Alternatively, the formulations disclosed herein may be administered intravenously, subcutaneously or intramuscularly. These formulations may be prepared by conventional means, and if necessary, the compositions may be mixed with any conventional additives, such as excipients, solubilizers, suspension aids, emulsifiers or preservatives. The disclosed excipients may perform multiple functions. For example, a solubilizer may also be a suspension aid, emulsifier, preservative, etc.

[0119] The present pharmaceutical composition can be conveniently provided in unit dosage form and can be prepared by any method well known in the pharmaceutical art. The amount of the composition that can be combined with a carrier material to produce a single dose will vary depending on the subject being treated and the particular mode of administration.

[0120] Methods for preparing these pharmaceutical compositions include the step of associating a carrier and, optionally, one or more accessory ingredients with the compositions of the present disclosure. In general, the pharmaceutical compositions are prepared by intimately and uniformly associating a liquid carrier with the drug.

[0121] It will be appreciated that the disclosed compositions can include the lyophilized or lyophilizing compounds disclosed herein. For example, compositions in the crystalline and / or amorphous powder form of the disclosed compounds are disclosed herein. Such forms can be reconstituted, for example, for use as aqueous compositions.

[0122] Injectable liquid dosage forms include pharmaceutically acceptable solutions, emulsions, microemulsions, solutions and suspensions. In addition to the subject composition, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, glycerol, tetrahydrofurfuryl alcohol and fatty acid esters of sorbitan, cyclodextrin, albumin, hyaluronic acid, chitosan and mixtures thereof. Polyethylene glycol (PEG) may be used to obtain desired properties such as solubility, stability, half-life and other pharmaceutically beneficial properties. Representative examples of stabilizing components include polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose and combinations thereof. Other excipients that may be used, such as solution binders or antioxidants, include butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methyl paraben, microcrystalline cellulose, polyvinylpyrrolidone, povidone, pregelatinized starch, propyl paraben, retinol palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C and xylitol, but are not limited thereto.

[0123] Typically, aqueous aerosols are made by formulating an aqueous solution or suspension of the subject composition together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular subject composition, but generally include nonionic surfactants (Tweens, pluronics or polyethylene glycols), innocuous proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosol agents are generally prepared from isotonic solutions.

[0124] It should be noted that the excipients given as examples can have multiple functions. For example, a solubilizer can also be a suspending aid, an emulsifier, a preservative, etc.

[0125] Examples of suitable aqueous and non-aqueous carriers that can be used in 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. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0126] The compositions of the present invention are generally in a sterile state, storage stable, and pharmaceutically acceptable, and can be easily administered as an aqueous solution without the need for reconstitution prior to administration. The compositions of the present invention are suitable for administration to a subject, which means that they are pharmaceutically acceptable, non-toxic, contain no components that would have an adverse effect on the biological effect of the chimeric nuclease, and have a pH close to that of a physiological state that avoids reactions at the inhalation and / or injection sites. The compositions of the present invention are, for example, cell-free.

[0127] The composition is generally stored in a sealed container, vial or cartridge that is generally suitable for long-term storage. "Suitable for long-term storage" means that the vial, container or cartridge does not cause leakage of the components of the composition of the present invention or intrusion of external components, such as microorganisms, when maintained at 25 °C for at least 3 months.

[0128] The composition of the present invention is preferably administered by spraying, generally by breath-activated spraying.

[0129] The composition of the present invention can also be administered by injection as described herein.

[0130] The composition 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.

[0131] A vial, as used herein, can also include two containers, one of which contains a lyophilized powder of a chimeric nuclease or lipid particles as described herein, and the second container contains a liquid for reconstitution of the lyophilized powder. The contents of the two containers can be mixed prior to administration.

[0132] As discussed above, the composition of the present invention can be administered by spraying. Suitable volumes of the composition of the present invention for spraying 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.

[0133] In the composition of the present invention, the concentration of the chimeric nuclease is from about 0.1 mg / ml to about 10.0 mg / ml, from about 10.0 mg / ml to about 100.0 mg / ml, from about 30.0 mg / ml to about 300.0 mg / ml, from about 500 mg / ml to about 2000 mg / ml and about 2.0 mg / ml.

[0134] In the composition of the present invention, the concentration of 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.

Claims

1. A polypeptide comprising the amino acid sequence of SEQ ID NO: 14 and comprising a substitution of aspartic acid to glutamic acid at the amino acid position corresponding to the 10th position of SEQ ID NO:

14.

2. A chimeric nuclease comprising a modified I-TevI nuclease domain, a linker, and an RNA-guided nuclease Staphylococcus aureus Cas9 comprising the amino acid sequence of SEQ ID NO: 14 and comprising a substitution of aspartic acid to glutamic acid at the amino acid position corresponding to the 10th position of SEQ ID NO: 14, wherein the modified I-TevI nuclease domain comprises SEQ ID NO:

6.

3. A pharmaceutically acceptable formulation comprising the chimeric nuclease according to claim 2, a guide RNA, lipid nanoparticles, and a pharmaceutically acceptable carrier thereof.

4. The formulation according to claim 3, wherein the lipid nanoparticles further comprise exogenous donor DNA.

5. The chimeric nuclease according to claim 2, which is used for editing genomic DNA of cells or organisms.

6. The formulation according to claim 3 or 4, wherein the formulation further comprises an excipient.

7. The formulation according to claim 6, wherein the excipient is selected from the group consisting of excipient 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.

8. Use of the formulation according to claim 3 or 4 in the manufacture of a medicament for treating a lung-related disease in a patient in need thereof.

9. The use according to claim 8, wherein the disease is cystic fibrosis.

10. The use according to claim 8, wherein the chimeric nuclease targets and cleaves the CFTR gene.

11. The use according to claim 10, wherein the CFTR delta F508 mutation is cleaved from the CFTR gene.

12. The use according to claim 8, wherein the disease is cancer.

13. The use according to claim 12, wherein the chimeric nuclease targets and cleaves the EGFR gene.

14. The use according to claim 12, wherein the cancer is non-small cell lung cancer.

15. The chimeric nuclease according to claim 2, wherein the linker is selected from the group consisting of SEQ ID NOs: 7 to 12.

16. The chimeric nuclease according to claim 2, comprising a guide RNA comprising the RNA sequence of SEQ ID NO:

15.

17. The chimeric nuclease according to claim 2, comprising a guide RNA comprising the RNA sequence of SEQ ID NO:

21.

18. The chimeric nuclease according to claim 2, comprising a guide RNA comprising the entire RNA sequence of SEQ ID NO:

16.

19. The chimeric nuclease according to claim 2, wherein the linker comprises one or more of the group consisting of SEQ ID NOs: 9 to 12.

20. The chimeric nuclease according to claim 2, wherein the RNA-guided nuclease Staphylococcus aureus Cas9 contains an Ala ５８０又は Ala ５５７ mutation.

Images