Lipid-encapsulated double-cut endonucleases for DNA and gene editing

Abstract

To provide a method for gene editing in which a chimeric nuclease is delivered to a cell or an organism without employing a viral vector.SOLUTION: Disclosed is a polypeptide comprising a complete specific amino acid sequence or a fragment thereof, where the fragment comprises a Glu10 mutation.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Array List This application contains a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. A copy of the above ASCII file, created on September 17, 2018, is Sequence_Listing_ST25.txt and is 77KB in size. [Background technology]

[0002] There are an estimated 5,000 to 10,000 monogenic disorders, defined as genetic conditions resulting from mutations in a single gene. These disorders often manifest during childhood, leading to a variety of conditions and sometimes resulting in early death. Together, they are estimated to affect approximately 6% of people at some point in their lives. Diagnosis and treatment for these disorders remain inadequate, and care is primarily symptomatic, focusing on disease management rather than addressing the underlying genetic deletion. There are also many other disorders in which mutations in a gene contribute to the pathogenesis of the disease.

[0003] Gene editing is a gene therapy approach that relies on designer nucleases to recognize and cleave specific DNA sequences, followed by the use of innate cellular DNA repair pathways, namely non-homologous end ligation (NHEJ) and homologous recombination repair (HDR), to introduce targeted modifications into the genome. Four nuclease families—meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided Cas9 (CRISPR-Cas9) nucleases associated with clustered and regularly arranged short palindromic sequence repeats—have been used in this regard. These can be designed to precisely introduce double-strand disruption at target loci of interest. Gene editing expands the possibilities for permanently modifying genomic sequences of interest by enabling targeted disruption, insertion, cleavage, and modification 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 modification efficiency, safety concerns related to nuclease specificity, and the 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 innate 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 altering the sequence of Cas9-associated guide RNA to be complementary to the target site. The ease of programming Cas9 targeting is in contrast to the more intensive protein modification required for other reagents (zinc finger nucleases (ZFNs), meganucleases, and activator-like effector nucleases (TALENs)). Along with proteins derived from the type III CRISPR system, Cas9 has been used for countless 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 resulting from mutations in the CFTR gene, which encodes an epithelial anion channel. The CFTR protein, a transmembrane conductance regulator in cystic fibrosis, is found in a wide range of organs, including the pancreas, kidneys, liver, lungs, gastrointestinal tract, and reproductive organs, thus making CF a multi-organ disease. Mutations in CFTR lead to suboptimal ion transport and fluid retention, causing prominent clinical symptoms such as abnormal mucus thickening in the lungs and pancreatic dysfunction. In the lungs, dysfunctional CFTR interferes with mucociliary clearance, making the organ susceptible to bacterial infection and inflammation, ultimately leading to airway obstruction, respiratory failure, and premature death. CF remains the most common and fatal genetic disorder in the Caucasian population, with an estimated 70,000–100,000 affected individuals worldwide, highlighting the urgent need for the development of better treatments.

[0006] One major challenge in developing therapeutic strategies for CF is the wide variety of mutation types. While the delta-F508 (deletion of phenylalanine at codon 508) mutation is by far the most common, prevalent in >80% of CF patients, over 1,990 harmful CFTR mutations have been described. These mutations result in dysfunctional CFTR with interrupted codons, abnormal splicing, incorrect protein folding or transport to the cell surface, and limited channel opening. Pharmacological interventions target some of these processes, and while drug administration is therapeutic for some gating mutation types, the common delta-F508 still requires more effective treatment. However, pharmaceutical advances in CF care have not addressed mutations resulting from abnormal splicing or interrupted codons; in these cases, gene editing may prove to be the most advantageous.

[0007] Similarly, in Western populations, approximately 15% of non-small cell lung cancer (NSCLC) patients have activating mutations in the EGF receptor (EGFR) gene within their tumors.

[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. Further, 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, immunogenicity after treatment, and genotoxicity. There is currently no non-viral delivery method for safely delivering nucleases to target cells and enabling controlled administration of nucleases in vivo.

[0009] The need for improvements to the existing gene editing technologies to address the above concerns to make gene editing technologies more efficient and effective is unmet.

Summary of the Invention

[0010] The present invention preferably lacks Met 1 and has Lys 26 (in the non-truncated version of I-TevI, Lys 27 ) and / or Cys 39 (in the non-truncated version of I-TevI, 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 140It contains one or more of the following, which may be wild-type or modified versions, preferably the Glu 10 Or Ala 557 A chimeric nuclease comprising a modified RNA-guided nuclease containing a mutation, Staphylococcus aureus Cas9 (the I-TevI ​​polypeptide comprising the entire amino acid sequence of SEQ ID NO: 6 or fragments thereof), and a guide RNA targeting the Cas9 domain, particularly SEQ ID NOs: 15, 16, 21 or fragments thereof; and a chimeric nuclease comprising a cationic and / or neutral lipid nanoparticle and optionally a DNA-binding compound, particularly GL67(N 4 This applies to pharmaceutically acceptable formulations containing (cholesteryl-spermine) and a pharmaceutically acceptable carrier thereof.

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

[0012] Another embodiment of the present invention relates to a method for editing genes by administering a chimeric nuclease to cells or organisms without using a viral vector, by using controlled administration in vivo.

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

[0014] In another embodiment, the novel chimeric nuclease targets two independent target sites on a selected DNA molecule, cleaving at either one or both target sites to produce a fragment 30-36 nucleotides long.

[0015] In a further example, novel purified chimeric nucleases contain additional guide RNA.

[0016] Another aspect of the present invention is the use of an extrusion process 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 a mixture 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 needing 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 comprising SEQ ID NOs: 7-12 or a fragment thereof, a linear single-stranded DNA comprising a homologous region adjacent to the site targeted and / or cleaved by a chimeric nuclease, a linear double-stranded DNA comprising a homologous region adjacent to the site targeted and / or cleaved by a chimeric nuclease, a double-stranded DNA of the same length comprising complementary DNA ends for that cleaved by a chimeric nuclease, a circular double-stranded DNA comprising a homologous region adjacent to the site targeted and / or cleaved by a chimeric nuclease, and a circular double-stranded DNA comprising an I-TevI target site and a Cas9 target site, wherein the product cleaved from the double-stranded DNA contains a complementary end to the end 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 further embodiments, the present invention comprises 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 LAGLIDADG, His-Cys Box, HNH, PD-(D / E)xK, and Vsr-like meganuclease, zinc-finger nuclease, scCas9 (Streptococcus canis), fnCas9 (Francisella novicida), cjCas9 (Campylobacter jejuni), Cpf1 (Lachnospiraceae bacterium), Cas12a (Acidaminococcus Sp (Acidaminococcus) The CRISPR protein selected from the group consisting of Sp)), Cas13a (Leptorichia shahii) and Cas3 (Streptococcus thermophilus), and the DNA-binding domain selected from the group consisting of a zinc-finger motif and a TALE activator domain.

[0023] In further examples, the present invention encompasses a modified RNA-guided nuclease Staphylococcus aureus Cas9 and guide RNA, wherein the guide RNA includes a sequence targeting a genetic polymorphism, a different sequence in the CFTR or EGFR gene, a sequence retargeting the nuclease, a cross-linked nucleic acid, and / or a mixture of guide RNAs. [Brief explanation of the drawing]

[0024] [Figure 1]Figure 1 is a schematic diagram of the prepared lipid-encapsulated double-cleavage nuclease (TevCas9) [components are not scaled]. The I-TevI ​​domain 10 is linked to the RNA-guided nuclease (Cas9) domain 12 via the linker domain 11. In a preferred embodiment, the formed particles also contain guide RNA 13 and donor DNA 14. The nuclease described above is contained in lipid particles 15 formed into a spherical shape using an extrusion process. [Figure 2] Figure 2 is a diagram illustrating the mechanism by which lipid-encapsulated TevCas9 is translocated into the cell and nucleus to reach its target DNA. As illustrated in Figure 2A, one or more cells 20 are exposed to a novel lipid-encapsulated nuclease particle 21 containing TevCas9 25, either in vivo or ex vivo. As shown in Figure 2B, the lipid-encapsulated nuclease particle 21 is endocytized into the cell 20. Endosomes 22 undergo maturation processes and move to the cytoplasm, where they are targeted for degradation (Figure 2C). In some cases, TevCas9 25 can evade endosomes 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 ("NLS"). As shown in Figure 2E, through its nuclear localization sequence, TevCas9 25 can enter the nucleus 23, and while in the nucleus 23, TevCas9 nuclease 25 binds to and cleaves the target genomic DNA sequence 24. [Figure 3]Figure 3 is a diagram illustrating the mechanism by which lipid-encapsulated TevCas9 modifies target DNA. The I-TevI ​​domain 27 targets the I-TevI ​​target sequence 29. The linker domain 30 ligates the Cas9 domain 28, which targets the Cas9 target sequence 31, with the I-TevI ​​domain 27. The gene mutation 32 is surrounded by or very close 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 predictable-sized deletion product 34 with non-complementary DNA ends 35, 36. Figure 3C illustrates that, in the presence of single-stranded donor DNA 37 with homologous arms, cell 20 can insert the donor DNA 37 sequence near the cleavage site via the homologous recombination repair (HDR) pathway 38. Figure 3D illustrates that, in the presence of donor DNA 39 having compatible DNA ends for those cleaved by TevCas9 25, cell 20 can insert the donor DNA sequence 39 between cleavage sites via directed ligation using the non-homologous end junction (NHEJ) pathway 40. In the absence of donor DNA, cell 20 can ligate DNA ends via the NHEJ pathway 40 (Figure 3E). [Figure 4] Figure 4A demonstrates that TevCas9, targeted to the CFTR gene using the guide in Sequence ID No. 15, cleaves the CFTR DNA substrate in vitro. Figure 4B shows cells transplanted with a plasmid DNA version of TevCas9 fused to a cleavable GFP tag, imaged 48 hours after treatment using phase contrast and GFP imaging on Cytation 5 (Biotek Instruments Inc, VT, USA). Genomic DNA was extracted from the recovered cells, and editing at the CFTR gene was detected by PCR amplification and T7 endonuclease I cleavage assay. [Figure 5] Figure 5 shows that TevCas9, targeted to the CFTR delta F508 mutation using the guide in Sequence ID No. 21, cleaves DNA substrates containing the CFTR delta F508 mutation in vitro, but does not cleave substrates containing the wild-type CFTR sequence. [Figure 6] Figure 6A illustrates how the saCas9 D10E mutation slows down the conversion of nicked supercoiled DNA to linear DNA. Figure 6B shows that on a linear EMX1 DNA substrate, the saCas9D10E(D10E) ribonucleoprotein complex (RNP) cleaves the target substrate at a level comparable to that of wild-type saCas9 (WT). The computer-predicted level of off-target editing by SaCas9D10E is lower than the level of off-target editing by wild-type saCas9. [Figure 7] Figure 7A is a schematic diagram of the I-TevI ​​site spacing in EGFR exon 19 deletion and wild-type (WT) EGFR. Figures 7B and 7C demonstrate that TevCas9 containing a nicking mutation in EGFR-targeted Cas9(H557A) using the guide RNA in Sequence ID No. 16 cleaves the EGFR exon 19 deletion DNA substrate four times faster than wild-type EGFR. Figure 7D shows that HCC827 cells with the EGFR exon 19 deletion mutation are selectively killed when treated with EGFR-targeted TevCas9 compared to NuLi-1 cells with wild-type EGFR (WT). [Modes for carrying out the invention]

[0025] Definitions and acronyms For convenience, certain terms used in this application, the examples, and the appended claims are summarized here. These definitions should be read in light of this disclosure and understood as those of a person skilled in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by a person skilled in the art.

[0026] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical objects of the articles. The terms “and / or” are defined herein as the possibility of having one or the other or both. For example, “A and / or B” provides a scenario having only A or only B or a combination of A and B. Where a claim indicates A and / or B and / or C, the composition may include as components 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 A, B and C.

[0027] The term “bioavailable” is recognized in the art and refers to forms of the disclosure in which it or a portion of the amount administered is absorbed, incorporated into, or otherwise made physiologically available to the subject or patient to whom it is administered.

[0028] When used herein, the term "exogenous donor DNA" refers to any sequence of DNA that is not, in whole or in part, identical to the original target DNA sequence.

[0029] The term "flexible linker," as used herein, refers to a situation in which the amino acid linker domain ensures the mobility of the I-TevI ​​domain to enable recognition, binding, and cleavage of the target DNA sequence under cellular physiological conditions when the RNA-guided nuclease domain (Cas9) binds to that target DNA sequence (typically: pH ~7.2, temperature ~37°C, [K+] ~140 mM, [Na+] ~5-15 mM, [Cl-] ~4 mM, [Ca++] ~0.0001 mM). The length of the amino acid linker may affect the number of nucleotides preferred between the Cas9 target site and the I-TevI ​​target site. Certain amino acids in the linker may also make specific contact with the DNA sequence targeted by TevCas9. These linker-DNA contacts may affect the flexibility of the I-TevI ​​domain. Amino acid substitutions in the linker domain may affect the linker domain's ability to contact DNA.

[0030] As used herein, the term "including" means "including but not limited to." "Including" and "including but not limited to" are interchangeable.

[0031] The terms “inhalation administration,” “inhaling,” “being inhaled,” “inhalation,” or “inhalation therapy,” which may be used interchangeably and as used herein, include the administration of a substantially uniform distribution of appropriately sized particles into the airway epithelium of the nose, central airways, peripheral lungs and / or alveolar regions of the lungs, or by intratracheal infusion. Such particles may be introduced and / or generated in a patient using appropriate equipment, preferably a sprayer.

[0032] The terms “patient,” “subject,” or “host” used in this method may refer to either a human or a non-human animal. Non-human animals include companion animals (e.g., cats and dogs) and animals raised for food (i.e., food animals), such as cattle, 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 is not harmful to the patient. Some examples of materials that can act as pharmaceutically acceptable carriers include: (1) sugars, e.g., dextrose, lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., microcrystalline cellulose, sodium carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose (HPMC), and cellulose acetate; (4) glycols, e.g., propylene glycol; (5) polyols, e.g., glycerol Phosphorus, sorbitol, mannitol, and polyethylene glycol; (6) esters, e.g., ethyl oleate, glyceryl behenate, and ethyl laurate; (7) buffers, e.g., mono- and di-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 suitable substances used in pharmaceutical formulations. The disclosed excipients may perform multiple functions. For example, solubilizers may also be suspension aids, emulsifiers, preservatives, etc.

[0034] In certain preferred embodiments, the pharmaceutically acceptable excipient is a crystalline bulking excipient. The terms “crystalline bulking excipient” or “crystalline bulking agent,” as used herein, mean an excipient that imparts bulk and structure to the lyophilized cake. These crystalline bulking agents are inert and do not react with proteins or nucleic acids. Furthermore, crystalline bulking agents can crystallize under lyophilized conditions. Examples of suitable crystalline fillers include hydrophilic excipients, e.g., water-soluble polymers; sugars, e.g., mannitol, sorbitol, xylitol, glucitol, ducitol, inositol, arabinitol, arabitol, galactitol, iditol, allitol, maltitol, fructose, sorbose, glucose, xylose, trehalose, allose, dextrose, altrose, lactose, glucose, fructose, glucose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, sucrose, maltose, lactose. Examples include tose, lactulose, fucose, rhamnose, melegitose, maltotriose, raffinose, althritol, their optically active forms (D- or L-type) and corresponding racemates; inorganic salts, both minerals and mineral organic compounds, such as calcium salts, such as lactates, glucons, glyceryl phosphates, citrates, mononucleotides and dinucleotides of phosphate, succinates, sulfates and tartrates, and the same salts of aluminum and magnesium; carbohydrates, such as conventional monosaccharides and disaccharides and 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. Dextran is a particularly useful bulking agent.

[0035] Where used herein, the term “pharmaceutically acceptable salt” refers to relatively non-toxic inorganic and organic acid addition salts or inorganic or organic base addition salts of a compound that are recognized in the art and include, for example, 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 with a different amino acid. As used herein, the abbreviation X10Y indicates that amino acid X, found at position 10 of the sequence, is substituted with amino acid Y. For example, W26C indicates that the amino acid tryptophan-26 (Trp, W) is replaced with cysteine ​​(Cys). Similarly, notation AA... X This indicates that AA is an amino acid that replaces the amino acid found at position X. For example, Lys 26 This indicates a substitution of the amino acid at position 26 of the sequence with lysine. Either abbreviation is interchangeable. Furthermore, the use of one-letter or three-letter abbreviations for amino acids is also interchangeable.

[0037] Where used herein, the term “therapeutic agent” refers to any chemical or biochemical part that is recognized in the art and is biologically, physiologically, or pharmacologically active in a subject, acting locally or systemically. Examples of therapeutic agents, also called “drugs,” are listed in well-known literature such as the Merck Index, the Physician's Desk Reference, and The Pharmacological Basis of Therapeutics, and include, without limitation, pharmaceuticals; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure, or alleviation of diseases or illnesses; 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] Where used herein, the term “therapeutic effect” refers to a topical or systemic effect, as recognized in the art and induced by a pharmacologically active substance, in animals, particularly mammals, and especially in humans. Accordingly, the term means any substance intended for use in the diagnosis, treatment, mitigation, management or prevention of disease, or in the promotion of a desired physical or mental manifestation and / or state in animals or humans. The phrase “therapeutic effective dose” means the amount of such substance that produces some desired topical or systemic effect with a theoretical benefit-risk ratio applicable to any treatment. The therapeutic effective dose of such a substance varies depending on the subject and disease state being treated, the subject’s weight and age, the severity of the disease state, the method of administration, etc., which can be readily determined by those skilled in the art. For example, a certain composition of the present invention may be administered in an amount sufficient to produce a theoretical benefit-risk ratio applicable to such treatment.

[0039] As used herein, the term “treating” includes any effect, such as improving, alleviating, reducing, regulating, or eliminating a condition, disease, or disorder. As used herein, “treating” may include both prophylactic and therapeutic treatments. For example, therapeutic treatments may include delaying, inhibiting, or preventing the progression of cystic fibrosis or non-small cell lung cancer, or alleviating or eliminating symptoms associated with cystic fibrosis or non-small cell lung cancer. Prophylactic treatments may 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 the 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] When used herein, “buffer solution” is any combination of acid or salt that is pharmaceutically acceptable and capable of maintaining the composition of the present invention within a desired pH range. The buffer solution 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 buffer solutions include any pharmaceutically acceptable buffer solution capable of maintaining the above pH range, such as acetic acid, tartaric acid, phosphoric acid, or citrate buffer. In one embodiment, the buffer solution is a phosphate buffer. In another embodiment, the buffer solution is an acetate buffer. In one embodiment, the buffer solution is disodium hydrogen phosphate, sodium chloride, potassium chloride, and potassium phosphate monobase.

[0042] In the disclosed compositions, the concentration of the buffer solution is generally in the range of 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, “antibiotic agent” is a pharmaceutically acceptable preservative suitable for administration to a subject in the compositions of the present invention, which inhibits, prevents, or delays the growth or growth of microorganisms, including, for example, bacteria, viruses, and fungi. 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, thiomersal, and nitric acid and phenylmercury acetate. In one embodiment, the antimicrobial agent is m-cresol, chlorocresol, or phenol. In another embodiment, the antimicrobial agent is chlorocresol or phenol. In yet another embodiment, the antimicrobial agent is phenol.

[0044] As used herein, an effective amount of an antimicrobial agent is an amount effective in the composition of the present invention to inhibit, prevent, or delay the growth or growth of microorganisms, including, for example, bacteria, viruses, and fungi. In the composition of the present invention, the amount of 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 may also be stored as powders that can be freeze-dried using freeze-drying techniques known in the art and reconstituted before administration. The term “freeze-drying,” as used herein, refers to freeze-drying or dehydration techniques that involve removing a solvent, preferably a water-miscible solvent, more preferably water, from a composition or the present invention by sublimation under high vacuum while the composition is frozen. Freeze-drying is generally carried out in a freeze-drying apparatus (freeze dryer), which includes a drying chamber with variable temperature control, a condenser for recovering water, and a vacuum system for reducing pressure in the drying chamber.

[0046] The term "lyophilized composition," as used herein, means any solid residue or powder produced or remaining after the lyophilized procedure described above. The lyophilized compositions of the present invention generally further comprise pharmaceutically acceptable excipients. The term "pharmaceutically acceptable excipient," as used herein, means any substance added to the solution before lyophilization to enhance the characteristics of the lyophilized cake, such as color, texture, strength, and volume. Pharmaceutically acceptable excipients may include, for example, buffers and pH adjusters, crystalline volume-increasing excipients, stabilizers, and tonicity-raising agents.

[0047] As used herein, a stabilizer is a composition that maintains the chemical, biological, or stability of a chimeric nuclease. Examples of stabilizers include polyols, which include sugars, 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 one that is suitable for administration to a subject, such as a human. The chimeranucleases 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 ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and organic bases, such as those derived from inorganic bases, such as alkoxides, alkylamides, alkyls, and arylamines. Therefore, useful bases for preparing the salts of the present invention include sodium hydroxide, potassium hydroxide, ammonium hydroxide, and potassium carbonate. Chimeranucleases of the present invention having sufficiently basic groups, such as amines, can react with organic or inorganic acids to form acid addition salts. Acids commonly used to form acid addition salts from compounds with basic groups include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. Examples of such salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propioates, oxalates, malons, succinates, suberates, sebacinates, fumarates, maleates, butyn-1,4-geoate, and hexyn-1 Examples include 6-geoates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoic acid, methoxybenzoates, phthalates, sulfonates, xylene sulfonates, phenyl acetate, phenylpropionate, phenyl butyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelates.

[0049] While specific embodiments of the present invention have been discussed, the above specification is illustrative and not limiting. Many variations of the present invention will become apparent to those skilled in the art upon reviewing this specification. The complete scope of the present invention can be determined by referring to the claims, along with such variations, as well as their equivalents and their entire scope in this specification.

[0050] Unless otherwise indicated, all numbers expressed herein and in the claims, such as component amounts and reaction conditions, are understood to be modified in all examples by the term "approximately." Accordingly, unless otherwise indicated, the numerical parameters expressed herein and in the appended claims are approximations that may vary depending on the desired properties to be obtained by the present invention.

[0051] The above discussion is intended to illustrate the principles and various embodiments of the present invention. With a full understanding of the above disclosure, the variations and modifications of the numerical values ​​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] abbreviation The abbreviations used herein are defined as follows: AA amino acids Cas9 CRISPR-related protein 9 CF (Cystic Fibrosis) CFTR (Cystic Fibrosis Transmembrane Conductance Regulatory Factor) gene cjCas9 Campylobacter jejuni Cas9 CRISPR from Cpf1 Prevotella and Francisella 1 CRISPR clustered, regularly arranged short palindromic repeats DLS (Dynamic Light Scattering) DMEM Dulbecco's Modified Eagle Medium DMPE 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine DNA (Deoxyribonucleic Acid) DOAB Dioctadecyldimethylammonium bromide DOPE 1,2-Dioleoyl-sn-Glycerol-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 homologous recombination restoration 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 nucleases TEV Tobacco Sex Virus TevCas9 modified I-TevI ​​domain, linker peptide, and modified RNA-guided nuclease Staphylococcus aureus Cas9 ZFN Zinc-Finger Nuclease

[0053] The inventors have 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 ("saCas9") (hereinafter referred to herein as "TevCas9"), which, when mixed with lipid nanoparticles with or without exogenous donor DNA, delivers to cells and, in the presence or absence of exogenous donor DNA, replaces DNA sequences in the presence of exogenous donor DNA or deletes DNA of a predetermined length in the absence of exogenous donor DNA. The novel chimeric nuclease has been shown to edit genes in both whole organisms (in vivo) and isolated cell cultures (ex vivo), not only in human cells but also in cells of other organisms such as bacteria, yeast, insects, plants, or other mammals.

[0054] The novel chimeric nuclease discovered by the inventors surpasses existing gene editing technologies and methods, particularly in the following advantages. a. A modified version of the TevCas9 nuclease can target two independent target sites as a single protein and 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, linker domain, Cas9 domain, or guide RNA (which targets the Cas9 domain to its target sequence). b. When a nuclease cleaves at two sites, it excises DNA of a precise length (~30-36 bases, depending on the sites targeted by I-TevI ​​and Cas9); The c.Cas9 domain contains a mutation (D10E) that is rationally designed to modify the Cas9 nuclease activity and / or improve the specificity of the Cas9 domain to its target binding site. d. In the presence of exogenous donor DNA, the present invention is designed to replace the target DNA sequence in a higher cellular percentage than existing techniques or practices. e. Nucleases can be purified as a single neighboring protein combined with guide RNA, which simplifies production; f. Lipid nanoparticles enable non-viral delivery to target cells with high efficiency and low toxicity, and allow for controlled administration of nucleases. While other lipid-based nuclease delivery technologies exist, none have compositions suitable for in vivo use; g. Lipid nanoparticles are also designed for nuclease delivery via spray (inhalation); One version of h. 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] Fusion of GIY-YIG nucleases, such as I-TevI, through a flexible linker to a DNA-binding domain is known (International Publication No. 2014 / 121222). A previous version of double-cleaved TevCas9 has been described, which includes amino acids 1-92 of the wild-type I-TevI ​​nuclease domain, a linker region containing amino acids 93-169 of the I-TevI ​​linker region, and Streptococcus pyogenes Cas9 ("spCas9") (Wolfs JM et al., (2016), 'Biasing Genome-Editing Events Toward Precise Length Deletions with an RNA-Guided TevCas9 Dual Nuclease,' Proc Natl Acad Sci USA, 113(52):14988-93). The chimeric nucleases of the present invention include the following: i. I-TevI ​​nuclease domains that bind novel target sequences, enabling targeting of clinically relevant gene sequences such as the CFTR gene; ii. Various flexible linker regions intended to impart different DNA binding or nuclease activity to TevCas9; iii. 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, compared to ~4.6 kilobases for Tev-spCas9) and a lower molecular weight TevCas9 protein (~144 kilodaltons for Tev-saCas9, compared to ~179 kilodaltons for Tev-spCas9), which is more suitable for multiple delivery techniques; cleavage by the saCas9 domain between the third and fourth nucleotides is more predictable compared to spCas9, as discovered by the inventors of the claimed techniques, and is more suitable for deletions of a defined length. iv. One version in which the guide RNA targets a specific CFTR gene sequence near the CFTR delta F508 mutation; and v. A second version in which the guide RNA is intended to target a specific EGFR gene sequence and cleave only DNA containing appropriately detached I-TevI ​​and Cas9 target sites. Such appropriately detached 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 comprises 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. Pharmaceutical formulations of lipids, nucleases, and exogenous donor DNA; c. Pharmaceutical formulations of lipids, nucleases, and exogenous donor DNA suitable for spraying (inhalation); and d. A version of the present invention comprising exogenous donor DNA that can be incorporated into a region between or around two sites targeted by a nuclease when delivered together with the TevCas9 nuclease in lipid nanoparticles.

[0056] The novel chimeric nuclease compositions of this application contain different combinations of I-TevI ​​domain, linker domain, Cas9 domain, and guide RNA.

[0057] The version targeting the CFTR gene consists of the following: i. The I-TevI ​​domain in the amino acid sequence according to Sequence ID No. 6; ii. A linker domain provided by any one of sequence numbers 7-12; iii. The saCas9 domain of the amino acid sequence according to SEQ ID NO: 13; and iv. Guide RNA for RNA sequences according to SEQ ID NO: 15 or 21.

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

[0059] A preferred embodiment of the I-TevI ​​domain is the 93-amino acid I-TevI ​​domain of the intestinal bacterium phage T4 with the following sequence: MGKSGIYQIKNTLNNKVYVGSAKDFEKRWKRHFKDLEKGCHSSIKLQRSFNKHGNVFECSILEEIPYEKDLIIERENFWIKELNSKINGYNIA(Sequence ID 6)

[0060] A preferred embodiment of saCas9 is a polypeptide consisting of 1,053 amino acids with the following sequence:

[0061] Glu in a preferred embodiment 10 The mutated saCas9 is a polypeptide consisting of 1,053 amino acids in the following sequence (mutations are underlined): MKRNYILGL E

[0062] The guide RNA for the CFTR gene-targeting version consists of 101 ribonucleotides with the following sequence: GCGUCAUCAAAGCAUGCCAACGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU(Sequence ID 15) AUAUCAUUGGUGUUUCCUAUGGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU(Sequence ID 21)

[0063] The guide RNA for the EGFR gene-targeting version is 101 ribonucleotides long with the following sequence: AAUUUUAACUUUCUCACCUUCGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUU(Sequence code 16).

[0064] The linker used in any of the above constructs may be selected from the following group: [Table 1]

[0065] synthesis Example 1: Method for producing TevCas9 nuclease: The DNA coding sequences of the I-TevI ​​domain, linker domain, and Cas9 domain described above were synthesized as a single adjacent 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 were synthesized. The oligonucleotides were mixed together in approximately 1 kilobase blocks of the sequences to be synthesized, and these ~1 kilobase blocks were synthesized using polymerase chain reaction (PCR). These ~1 kilobase blocks were then mixed and subjected to PCR to synthesize the I-TevI ​​domain, linker domain, and Cas9 domain. Furthermore, the TevCas9 DNA sequence was optimized before synthesis to promote TevCas9 expression in E. coli and to simplify restriction enzyme digestion. First, less frequently used 3-base pair DNA codons in Escherichia coli ("E. coli") were replaced with more frequently occurring ones (for example, the relative abundance of codon AGG in the 6 codons encoding the amino acid arginine was 0.03 compared to 0.42 for codon CGT). In total, 37% of the codons were changed to those more favorable to E. coli. Second, the content of the 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 adjacent DNA is digested with restriction endonucleases NdeI and BamHI (New England Biolabs, Ipswich, MA, United States), in which the target site appears only once in its DNA sequence. Then, using DNA ligase (New England Biolabs, Ipswich, MA, United States), it is inserted into a similarly digested pET-11a expression vector (EMD Millipore, Burlington, MA, United States) suitable for TevCas9 expression in E. coli.The pET-11a vector containing TevCas9 is used to transform 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, 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 at an optical density of 0.4-0.6 as measured by a spectrophotometer at a wavelength of 600 nM, and the expression of the 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. Successful TevCas9 expression is verified by the presence of a band of approximately 150 kDa on a Coomassi-stained SDS-polyacrylamide gel in the inducer sample compared to the non-inducible sample. E. coli cells are collected 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 sodium phosphate (dibasic) (Sigma, St. Louis, MO, United States), pH 8.0 [Buffer 1]. Alternatively, the sodium phosphate (dibasic) in Buffer 1 can be replaced with 10 mM Tris hydrochloride (Sigma, St. Louis, MO, United States), pH 8.E. coli is dissolved by homogenization using any other suitable dissolution method known in the art, such as high-pressure liquid operated at 600-1000 bar, homogenizer (Avestin Inc., Ottawa, ON, Canada), sonication using lysozyme treatment (Branson Ultrasonics Corp, Danbury, CT, United States), 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 dissolved substance is centrifuged at 12,000 rpm for 20-30 minutes at 4°C, and the supernatant containing soluble TevCas9 is used in subsequent purification steps. The pellet contains cell debris, insoluble intracellular material, and some insoluble TevCas9. The success and solubility of dissolution are verified by the presence of a band of approximately 150 kDa on the Coomassi-stained SDS-polyacrylamide gel in the supernatant sample compared to the resuspended pellet sample.

[0066] The TevCas9 nuclease is purified in the following steps: 1. The nuclease-containing solution is placed on an immobilized metal affinity chromatography (IMAC) column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) that binds to the nuclease. 2. Wash the IMAC column 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 to 500 mM sodium chloride (Sigma-Aldrich, St. Louis, MO, United States), and 50 mM dibasic sodium phosphate (Sigma, St. Louis, MO, United States), pH 7.6 to 8.0 [Buffer 2]. Alternatively, replace the dibasic sodium phosphate in Buffer 2 with 10 mM Tris hydrochloric acid (Sigma, St. Louis MO, United States), pH 7.6 to 8. 4. The eluted material is treated with tobacco etch virus (TEV) protease (New England Biolabs, Ipswich, MA, United States) and incubated with appropriate guide RNA. The guide RNA is synthesized by Integrated DNA Technology Inc. (Coralville, IA, United States). 5. The treated eluate is placed back onto the IMAC column, and the flow-through containing the TevCas9 nuclease and guide RNA is collected. 6. The success of TevCas9 nuclease purification is confirmed by the presence of a 150 kilodalton protein band on a Coomassi-stained SDS-polyacrylamide gel. The success of co-purification of TevCas9 with guide RNA is confirmed by treating the eluate sample with proteinase K (New England Biolabs, Ipswich, MA, United States). The sample is then divided into two parts; one subsample is further treated with RNase A (New England Biolabs, Ipswich, MA, United States), and the other is kept in a control buffer without RNase A. A ~100 nucleotide RNA band is visible on the urea-polyacrylamide gel in the control sample, but not in the RNase A-treated sample. 7. Dialysis is performed on the solution containing TevCas9 nuclease and guide RNA to a solution containing phosphate-buffered saline with a pH of 7.4.

[0067] Example 2: Method for producing lipid nanoparticles A lipid nanoparticle of a preferred embodiment consists of one of the following mixtures: I. Lipid nanoparticle number 1 contains DOPE (Avanti Polar Lipids, Alabaster, AL, United States) and MPEG-5000-DMPE (Avanti Polar Lipids, Alabaster, AL, United States) in a molar ratio of 2:0.05, respectively; II. Lipid nanoparticle number 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 number 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] Lipid nanoparticles are manufactured to have an average diameter of approximately 100 nM.

[0069] Select one of the lipid mixtures numbered 1 to 3. For example, mix DOPE and MPEG-5000-DMPE 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 filtered and sterilized through a 0.2 μM sterile filter (VWR Scientific, Radnor, PA, United States). The average diameter and particle 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 DNA sequences intended to repair genetic abnormalities. This includes DNA sequences not found in the target genomic DNA; these sequences are intended to introduce one or more DNA sequences that do not interfere with normal gene function but are used to knock out I-TevI ​​and / or Cas9 sites and / or to track the success of targeted gene repair. Examples of donor DNA include, but are not limited to, the following: Linear single-stranded DNA of various lengths, including homologous regions adjacent to the site targeted / cleaved by I.TevCas9. II. Linear double-stranded DNA of various lengths, including homologous regions adjacent to the sites targeted / cleaved by TevCas9; III. Double-stranded DNA of equal length, including DNA ends that are cleaved by a nuclease and also complementary to those cleaved by TevCas9; IV. Circular double-stranded DNA containing homologous regions adjacent to the sites targeted / cleaved by TevCas9; and V. A circular double-stranded DNA containing I-TevI ​​target sites and Cas9 target sites, wherein the product cleaved from the double-stranded DNA contains ends complementary to those cleaved by TevCas9.

[0071] Example 4: Method for assembling lipid-encapsulated TevCas9 and transferring genes into cells For ex vivo cell gene transfer: To assemble lipid-encapsulated TevCas9, lipid nanoparticles are mixed with TevCas9 in Dulbecco's Modified Eagle Medium (DMEM) (Sigma, St. Louis, MO, United States) at a molar ratio of 2000:1 and incubated at room temperature for 10 minutes. Gene transfer is performed into cells using 8.7 x 10⁻¹⁷ to 3.1 x 10⁻¹⁷ moles of lipid-encapsulated TevCas9 per cell.

[0072] For in vivo cell-to-cell gene transfer: To assemble lipid-encapsulated TevCas9, mix lipid nanoparticles with TevCas9 in phosphate-buffered saline at a molar ratio of 2000:1 and allow to stand at room temperature for 10 minutes. Determine the molar ratio of lipid-encapsulated TevCas9 / cell for in vivo gene transfer.

[0073] Other Embodiments Nucleases may contain different combinations of 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 may contain different combinations of mutations that alter the site targeted by the I-TevI ​​domain or the activity of the I-TevI ​​domain, including mutations that alter the sequence recognized by I-TevI, such as K26 and / or C39. Other versions of the nuclease may replace the I-TevI ​​domain with other GIY-YIG nuclease domains, such as I-BmoI and Eco29kI. Other versions may be expressed in E. coli as a result of treatment. 1 It does not contain [the specified ingredient].

[0075] Linker domain modification: The linker domain may include the following, and may also include one that alters 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 amino acids shown in SEQ ID NOs. 9-12.

[0076] Modifications to the Cas9 domain: Other versions of the Cas9 domain may include: a) a version of the saCas9 domain containing the D10E mutation (SEQ ID NO: 14); b) a version of the saCas9 domain that cleaves the target DNA on one strand of the target DNA, e.g., 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, e.g., mutations in both the D10A and H557A mutations (SEQ ID NO: 18); d) a previously described spCas9EQR variant containing the D1135E, R1335Q and T1337R mutations combined with the D10E mutation (SEQ ID NO: 19); and e) a previously described spCas9EQR variant containing the D1135E, R1335Q and T1337R mutations combined with the D10E mutation and a mutation that cleaves the target DNA on one strand of the target DNA, e.g., the H840A mutation (SEQ ID NO: 20). Other versions of the saCas9 domain include Met 1 It does not contain [the specified ingredient].

[0077] Other versions may involve substituting the Cas9 domain with other nuclease or DNA-binding domains, e.g., a) meganucleases, e.g., family LAGLIDADG, His-Cys Box, HNH, PD-(D / E)xK, Vsr-like, etc.; b) zinc-finger nucleases; c) other CRISPR proteins, e.g., scCas9, fnCas9, cjCas9, Cpf1, Cas12a, Cas13a, Cas3, etc.; and d) other DNA-binding domains, e.g., zinc-finger motifs, TALE activator domains, etc.

[0078] Modifications to guide RNA: a) Other versions of guide RNA may target the same region of DNA in the CFTR gene or EGFR gene but contain different sequences that constitute genetic polymorphisms in the population; b) Other versions of guide RNA may target different sequences in the CFTR gene or EGFR gene; c) Other versions of guide RNA may target other sequences in the genome to retarget nucleases to further clinically relevant targets; d) Other versions of guide RNA may contain cross-linked nucleic acids ("BNA") to enhance 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 GL67(N 4 f) The lipid nanoparticles may contain DNA-binding compounds such as cholesteryl-spermine; g) The lipid nanoparticles may be freeze-dried to enhance stability; and g) The lipid nanoparticles may be resuspended in solutions other than phosphate-buffered saline, such as sterile isotonic saline or water for injection.

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

[0081] Modifications to the method of assembling lipid-encapsulated nucleases and transferring genes into cells: a) Other versions of lipid-encapsulated nucleases may contain different molar ratios of lipid nanoparticles to nuclease; b) Culture media other than DMEM or phosphate-buffered saline may be used for the incubation stage; c) Nucleases and lipid nanoparticles may be incubated for less than 10 minutes or more than 10 minutes; and d) Other molar amounts of nuclease / cell may be used in the gene transfer reaction.

[0082] Modifications to the method for producing the nuclease: 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) may be used; b) Buffer 1 or 2 may contain different concentrations of imidazole, sodium chloride, dibasic sodium phosphate, or tris hydrochloride and be buffered to different pH levels; c) Other processing steps such as cation or anion exchange chromatography may be used; d) The nuclease may be dialyzed to a solution other than phosphate-buffered saline, such as sterile isotonic saline or water for injection; e) The nuclease may be lyophilized to enhance stability; f) Guide RNA may be co-expressed from a 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 endonuclease, 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: A method for demonstrating the modification of CFTR delta F508 and CFTR protein function in a model cell line. Immortalized epithelial cells homozygous for the CFTR delta-F508 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) from pharmaceutical formulations targeted to the CFTR delta-F508 mutation. Appropriate control cell lines, such as immortalized epithelial cells homozygous for wild-type CFTR (e.g., NuLi-1 (ATCC® CRL-4011®, American Type Culture Collection, Manassas, VA, United States)), are also used.

[0084] The proportion of cells with corrected CFTR delta F508 compared to uncorrected cells is measured by T7 endonuclease I assay (EnGen® Mutation Detection Kit, New England Biolabs #E3321, Ipswich, MA, United States), PCR-amplification target site restriction endonuclease digestion (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 method. The effect of TevCas9 treatment on a control cell line (e.g., NuLi-1 (ATCC® CRL-4011®, American Type Culture Collection, Manassas, VA, United States)) will be evaluated. CFTR functionality will be measured using short-circuit current measurement in a Ussing Camber (Warner Instruments, Hamden, CT, United States) in the presence of a chloride ion gradient, in both treated CuFi-1 cultures (ATCC® CRL-4013®, American Type Culture Collection, Manassas, VA, United States) and sham-treated CuFi-1 cultures (ATCC® CRL-4013®, American Type Culture Collection, Manassas, VA, United States). The effect of TevCas9 treatment on a control cell line (e.g., NuLi-1) will also be measured.

[0085] To demonstrate EGFR exon 19 deletion mutations and the 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 with lipid-encapsulated TevCas9 (Specific Biologics, Toronto, ON, Canada) in pharmaceutical formulations of phosphate-buffered saline, sterile isotonic saline, or water for injection, which are targeted to EGFR exon 19 deletion. Appropriate control cell lines, such as NuLi-1 (ATCC® CRL-4011®, American Type Culture Collection, Manassas, VA) or immortalized epithelial cell lines homozygous to wild-type EGFR, are used.

[0086] The percentage of cells with disrupted EGFR exon 19 deletion compared to uncorrected cells will be measured by T7 endonuclease I assay (EnGen® Mutation Detection Kit, New England Biolabs #E3321, Ipswich, MA, United States), restriction endonuclease digestion of the PCR amplification 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 will also be evaluated, along with 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)). The effects of TevCas9 treatment in a control cell line (e.g., NuLi-1 (ATCC® CRL-2868®, American Type Culture Collection, Manassas, VA, United States)) will be evaluated using an enzyme-linked immunosorbent assay (ELISA) (Sigma, St. Louis, MO, United States) to detect phosphorylated (i.e., activated), non-phosphorylated, and total EGFR proteins in treated HCC827 cultures versus sham-treated HCC827 cultures.

[0087] Example 1: Animal model study designed to demonstrate efficacy and determine dose-limiting toxicity In a method for demonstrating the modification of CFTR delta-F508 and / or cystic fibrosis symptoms using lipid-encapsulated TevCas9 treatment in animal models (e.g., mice, rats, miniature pigs, or ferrets), lipid-encapsulated TevCas9 targeted to CFTR delta-F508 is delivered directly to the lungs by either intubation or intranasal delivery in a pharmaceutical formulation of phosphate-buffered saline, sterile isotonic saline, or water for injection. The procedure time is approximately 30 to 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 sprayer (Aeroneb®, AeroEclipse®, (Trudell Medical, London, ON, Canada)) or PARI-LC Plus®, (PARI USA, Midlothian, VA, United States)). The average particle size of approximately 100 nM lipid nanoparticles is confirmed after spraying by dynamic light scattering (DLS) using a Zetasizer (Malvern Panalytical Ltd, Malvern, United Kingdom) or by another suitable technique known in the art. The composition and concentration of lipid-encapsulated TevCas9 are confirmed after spraying using a MicroGram Lipid Assay Kit (ProFoldin, Hudson, MA, United States) and the presence of an approximately 150 kDa band on a Coomassie-stained SDS-polyacrylamide gel. To measure the rate of gene modification, representative sheep (miniature pig) animal models homozygous for the CFTR delta-F508 mutation (Exemplar Genetics, Sioux City, IA, United States) will be exposed orally, nasally, or directly to the lungs with lipid-encapsulated TevCas9 targeted to CFTR delta-F508 and appropriate controls. The overall maintenance of these animals will include breeding and parturing; age-appropriate bio-secure housing; 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 will be treated with lipid-encapsulated TevCas9 concentrations predicted to be effective from the above model cell line experiments for 2 days to 4 weeks for acute toxicity studies and for up to 24 months for chronic toxicity studies.

[0089] The overall health status of the animals should be monitored post-treatment to assess all treatment-related adverse events, including changes in behavior, weight, or food consumption; immune response; changes in cardiovascular health; and mortality. Other indicators of post-treatment effectiveness may include: I. Mandatory respiration in each animal after treatment, e.g., forced expiratory volume (or other appropriate method); II. Overall survival of each animal compared to the control; III. Other indicators of lung function (e.g., use of a mechanical ventilator capable of performing general lung function assessment); and IV. Measurement of mutations in vivo through tissue sampling and mutation detection methods, such as polymerase chain reaction.

[0090] After treatment with lipid-encapsulated TevCas9, the animals are slaughtered and lung and tracheal tissue is collected.

[0091] The proportion of cells with modified CFTR delta F508 compared to unmodified cells is measured by T7 endonuclease I assay (EnGen® Mutation Detection Kit, New England Biolabs #E3321, Ipswich, MA, United States), restriction endonuclease digestion of PCR amplification target sites (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 method.

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

[0093] Alternatively, lipid-encapsulated TevCas9 targeted to EGFR exon 19 deletion mutations in a pharmaceutical formulation is sprayed using a commercially available sprayer ((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 other appropriate techniques known in the art. The composition and concentration of the lipid-encapsulated TevCas9 nanoparticles are confirmed using a MicroGram Lipid Assay Kit (ProFoldin, Hudson, MA, United). Confirmation is made after spraying using the presence of an approximately 150 kDa band on a Coomassi-stained SDS-polyacrylamide gel. To measure the ratio of gene disruption, representative murine (mouse) animal models homozygous for EGFR exon 19 deletion mutations are exposed to lipid-encapsulated TevCas9 targeted to EGFR exon 19 deletion via the nose, oral cavity, or direct lung exposure. Mice are treated with various concentrations of TevCas9 predicted to be effective from model cell line experiments for 2 days to 4 weeks for acute toxicity studies and up to 24 months for chronic toxicity studies.

[0094] The overall health status of the animals should be monitored post-treatment to assess all treatment-related adverse events, including changes in behavior, weight, or food consumption; immune response; changes in cardiovascular health; and mortality. Other indicators of post-treatment effectiveness may include: I. Quantification of EGFR-activating protein by positron emission tomography (PET) using EGFR mutant tracers; II. Overall survival of each animal compared to the control; and III. Measurement of tumor formation / reduction in each animal over time. In vivo evaluation of mutations through tissue sampling and mutation detection methods, such as the Cobas® EGFR mutation test version 2 (Roche Diagnostics, Risch-Rotkreuz, Switzerland). After treatment with spray lipid-encapsulated TevCas9, animals are sacrificed and lung and tracheal tissue is collected.

[0095] The proportion of cells with disrupted EGFR exon 19 deletion mutations compared to undisrupted cells is measured by T7 endonuclease I assay (EnGen® Mutation Detection Kit, New England Biolabs #E3321, Ipswich, MA, United States), restriction endonuclease digestion of PCR amplification target sites (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 method. EGFR protein expression and activity in cells of recovered tissue are measured using enzyme-linked immunosorbent assay (ELISA) (Sigma, St. Louis, MO, United States) to detect phosphorylated (i.e., activated), unphosphorylated, and total EGFR proteins. To enable the first clinical trials in humans, dose-limiting toxicity will be determined based on the expected effective dose from the animal model experiments discussed above. Various concentrations of lipid-encapsulated TevCas9 (e.g., milligrams / kilogram body weight) will be sprayed and delivered to suitable animal models for toxicity testing, such as cynomolgus monkeys or other non-human primates. The overall health status of the animals will be monitored for any treatment-related adverse events, including changes in behavior, body weight, or food consumption; immune responses; changes in cardiovascular health; and mortality. Other indicators of efficacy may be measured in this study, including those described above.

[0096] Therapeutic effect The novel chimeric nucleases of the present invention are intentionally designed to modify the DNA of lung epithelial cells to treat monogenic diseases, but they 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 replacement 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 uptake by the present invention. As illustrated in Figure 2A, one or more cells 20 are exposed to a novel lipid-encapsulated nuclease particle 21 containing TevCas9 25 by administration either in vivo or ex vivo. As shown in Figure 2B, the lipid-encapsulated nuclease particle 21 is endocytized into the cells 20. The endosomes 22 undergo a maturation process in the cytoplasm and are targeted for degradation (Figure 2C). Under certain circumstances, TevCas9 25 can evade the endosomes 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 ("NLS"). As shown in Figure 2E, through its nuclear localization sequence, TevCas9 25 can enter the nucleus 23, and while in the nucleus 23, the TevCas9 nuclease 25 binds to the target genomic DNA sequence 24 and cleaves it 26.

[0098] Figure 3 illustrates the mechanism of TevCas9 nuclease in DNA cleavage. Figure 3A is representative of the key properties of TevCas9 that bind 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 ligates the Cas9 domain 28, which targets the Cas9 target sequence 31, with the I-TevI ​​domain 27. The gene mutation 32 is surrounded by or adjacent 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 predictable deletion product 34 with non-complementary DNA ends 35, 36. Figure 3C illustrates that, in the presence of single-stranded donor DNA 37 with homologous arms, cell 20 may insert the donor DNA 37 sequence near the cleavage site via the homologous recombination repair (HDR) pathway 38. Figure 3D illustrates that, in the presence of donor DNA 39 with compatible DNA ends for cleavage by TevCas9 25, cell 20 may insert the donor DNA sequence 39 between the cleavage sites via directed ligation using the non-homologous end ligation (NHEJ) pathway 40. In the absence of donor DNA, cell 20 may ligate DNA ends via the NHEJ pathway 40 (Figure 3E).

[0099] Example 2: Treatment of cystic fibrosis In the treatment of cystic fibrosis, the exogenous donor DNA contains DNA sequences that repair the CFTR delta F508 mutation, which is involved in a method of targeted deletion of a predetermined length of DNA sequence in human somatic cells, in order to stimulate homologous recombination repair using the exogenous donor DNA as a template (Figure 3C).

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

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

[0102] The following examples are provided to illustrate methods for preparing and using the compounds disclosed herein, without limiting the scope of this disclosure. Many other embodiments of this disclosure will be apparent to those skilled in the art.

[0103] A nebulizer is a device that delivers drugs to the lungs in the form of aerosolized vapor. Nebulizers are commonly used to treat respiratory diseases such as asthma and COPD, for example, by spraying corticosteroids, but they have also been used to treat and prevent lung infections, such as ARIKAYCE® (Insmed Incorporated, Bridgewater, NJ, United States).

[0104] A nebulizer may require several steps to prepare the liquid for spraying. The drug is typically held in liquid form in a cup inside the nebulizer chamber. Once introduced, the device is switched on, generating compressed air to convert the liquid into vapor in the nebulizer chamber. The patient places the mouthpiece of the nebulizer chamber to their mouth, takes a sharp, deep inhale, and holds their breath for 5-10 seconds to ensure the drug reaches the lower part of the lungs. Various types of such devices exist. Many modern nebulizers are activated by respiration and depend on the patient's inhalation power to synchronize the aerosolized liquid from the device, thus ensuring the drug is delivered only to the patient and not to the surrounding environment. This also ensures consistent 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, including Aeroneb®, AeroEclipse® (Trudell Medical, London, ON, Canada) or PARI-LC Plus® (PARI USA, Midlothian, VA, United States). In embodiments of the present invention, a nebulizer is used for the delivery of the present invention's novel lipid-encapsulated chimeric nuclease containing 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 a 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 include: a lower amount of drug may be required compared to oral or intravenous administration; the onset of action may be faster via inhalation compared to oral routes; the severity of adverse effects may be lower because the drug is delivered locally to the lung tissue where the disease manifests itself; and inhaled drug therapy is painless and relatively comfortable for the patient, which promotes compliance.

[0107] There is no non-invasive delivery route that offers the same rate of action as inhaled drugs. One advantage of inhaled drugs is that they are absorbed more quickly than molecules injected subcutaneously, providing a more immediate physiological response. Small or large molecules, especially hydrophobic molecules, can be absorbed within seconds of inhalation and can therefore be used to treat a wide range of symptoms that occur suddenly or require long-term administration. Pain, panic, anxiety, nausea, acute cardiovascular onset, bronchoconstriction, sleep induction, spasms, Parkinson's lock-up, and hot flashes are some of the acute onset conditions that can be addressed with inhaled drugs.

[0108] Most protein-based drug products are somewhat water-soluble and are rapidly and effectively absorbed from the lungs. Higher hydrophobic substances are absorbed even more rapidly, within seconds to minutes. Higher hydrophilic substances are absorbed within minutes to 10 minutes. In one example of the present 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 containing a modified I-TevI ​​nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9, which is both water-soluble and hydrophilic. The dose can range from 1 to 1000 milligrams of lipid nanoparticles and chimeric nuclease, respectively, with about 5 to 200 milligrams being preferred. The claimed lipid-encapsulated chimeric nuclease, containing a modified I-TevI ​​nuclease domain, a linker, and a modified RNA-guided nuclease Staphylococcus aureus Cas9, was absorbed by lung cells within hours, and complete cleavage on DNA substrates in vitro was observed within 2 hours. Other spray therapeutic agents were delivered daily. Spray administration of the lipid-encapsulated chimeric nuclease may therefore be daily or less frequent, depending on its efficacy on a patient basis. 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 patient's symptoms, age and weight, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the composition. Any of the formulations may be administered as a single dose or in divided doses. The dosage of the composition can be readily determined by the art known to those skilled in the art or as taught herein.

[0110] In one embodiment, the dosage of the compound in question would generally be in the range of about 1 to 1000 milligrams, specifically about 5 to 200 milligrams, depending on the patient's body weight.

[0111] Any potential effects of the formulation on the effective dose or amount and timing of administration may need to be confirmed for any specific composition of this disclosure. This may be completed by conventional experiments as described herein, using one or more animal groups (preferably at least five animals per group), or, if necessary, in human clinical trials. The effectiveness of any target composition and method for treatment or prevention may be evaluated by administering the composition, measuring one or more applicable indicators, and comparing the post-treatment values ​​of these indicators with the pre-treatment values ​​of the same indicators to assess the effect of the administration.

[0112] The precise timing and amount of administration of a particular composition that will produce the most effective treatment in a given patient depends on the composition's activity, pharmacokinetics, and bioavailability, the patient's physiological state (including age, sex, disease type and stage, overall health status, and response to a particular dose and type of drug), and the route of administration. The guidelines provided herein may be used, for example, to determine the optimal time and / or dose to optimize treatment, which will require no more than routine experiments consisting of monitoring the subject and adjusting the dose and / or timing.

[0113] While the subject is being treated, the patient's health status can be monitored by measuring one or more relevant indicators at predetermined intervals during the treatment period. The treatment, including its composition, dosage, administration time, and formulation, can be optimized based on the results of such monitoring. The patient can be periodically re-evaluated 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 a dose lower than the optimal dose of this compound. The dose may then be gradually increased until the optimal therapeutic effect is achieved.

[0115] The use of the composition in question may reduce the required dosage for any individual drug contained in the composition, as the onset and duration of effects of different drugs may be complementary.

[0116] The therapeutic efficacy of the target composition is, for example, LD 50 and ED 50 This can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.

[0117] Data obtained from cell culture assays and animal experiments may be used when formulating dosage ranges for human use. The dosage of any target composition is preferably low-toxicity or non-toxic. 50 The concentration falls within the range of [specific component]. The dose may vary within this range depending on the dosage form used and the route of administration utilized. For the compositions of this disclosure, the therapeutically effective dose can first be estimated from a cell culture assay.

[0118] prescription The pharmaceutical compositions of this disclosure may be administered by various means depending on their intended use, as is well known in the art. For example, the compositions of this disclosure may be administered by spray. Alternatively, the formulations disclosed herein may be administered intravenously, subcutaneously, or intramuscularly. These formulations may be prepared by conventional means, and the compositions may, if necessary, be mixed with some 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] This formulation can be conveniently provided in unit dosage forms and can be prepared by any method well known in the field of pharmaceutical technology. The amount of composition that can be combined with a carrier material to produce a single dose varies depending on the subject being treated and the specific method of administration.

[0120] Methods for preparing these formulations include the step of associating a carrier and, optionally, one or more auxiliary components with the composition of the Disclosure. Generally, the formulations are prepared by homogeneously and densely associating a liquid carrier with a drug.

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

[0122] Liquid dosage forms for injection include pharmaceutically acceptable solutions, emulsions, microemulsions, solutions, and suspensions. In addition to the composition of interest, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, glycerol, fatty acid esters of tetrahydrofuryl alcohol and sorbitan, cyclodextrin, albumin, hyaluronic acid, chitosan, and mixtures thereof. Polyethylene glycol (PEG) may be used to obtain desired properties of 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, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (alpha-tocopherol), vitamin C, and xylitol.

[0123] Typically, aqueous aerosols are prepared by formulating an aqueous solution or suspension of the composition of interest together with conventionally pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary depending on the requirements of the specific composition of interest, but generally include nonionic surfactants (Tweens, pluronics, or polyethylene glycol), harmless proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars, or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

[0124] It is noteworthy that the excipients given as examples may have multiple functions. For example, a solubilizer may also function as a suspension aid, emulsifier, preservative, and so on.

[0125] Suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of this 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 by the use of coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

[0126] The compositions of the present invention generally allow for the easy administration of sterile, storage-stable, and pharmaceutically acceptable aqueous solutions without the need for reconstitution before administration. The compositions of the present invention are suitable for administration to subjects, meaning they are pharmaceutically acceptable, non-toxic, contain no components that adversely affect the biological effects of chimeric nucleases, and have a pH close to that of a physiological state to avoid inhalation and / or injection site reactions. The compositions of the present invention, for example, do not contain cells.

[0127] This composition is generally stored in a sealed container, vial, or cartridge suitable for long-term storage. "Suitable for long-term storage" means that the vial, container, or cartridge does not allow 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 spray, generally by a respiration-activated spray.

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

[0130] The compositions of the present invention may be administered alone or in combination with further therapeutic agents such as antiviral agents, antimicrobial agents, chemotherapeutic agents, and immunotherapies.

[0131] When used herein, the vial may also include two containers, one of which contains the chimeric nuclease or lipid particles of the lyophilized powder as described herein, and the second container containing a liquid for reconstituting the lyophilized powder. The contents of the two containers may be mixed before 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 about 0.5 to about 1 ml, about 1 to about 2 ml, about 2 to about 10 ml, or about 10 to about 20 ml.

[0133] In the compositions of the present invention, the concentrations of chimeric nuclease are approximately 0.1 mg / ml to approximately 10.0 mg / ml, approximately 10.0 mg / ml to approximately 100.0 mg / ml, approximately 30.0 mg / ml to approximately 300.0 mg / ml, approximately 500 mg / ml to approximately 2000 mg / ml, and approximately 2.0 mg / ml.

[0134] In the composition of the present invention, the concentrations of lipid nanoparticles are approximately 0.1 mg / ml to approximately 10.0 mg / ml, approximately 10.0 mg / ml to approximately 100.0 mg / ml, approximately 30.0 mg / ml to approximately 300.0 mg / ml, approximately 500 mg / ml to approximately 2000 mg / ml, and approximately 2.0 mg / ml.

Claims

1. A nuclease comprising modified Staphylococcus aureus Cas9, wherein the modified Staphylococcus aureus Cas9 comprises a substitution of aspartic acid to glutamic acid in the amino acid corresponding to the 10th position of SEQ ID NO: 13, and the amino acid sequence of Staphylococcus aureus Cas9 has at least 99% sequence identity with SEQ ID NO:

13.

2. The nuclease according to claim 1, wherein the modified Staphylococcus aureus Cas9 comprises a histidine to alanine substitution in the amino acid corresponding to position 557 of SEQ ID NO:

13.

3. The nuclease according to claim 1, further comprising an I-TevI ​​nuclease domain.

4. The nuclease according to claim 3, wherein the I-TevI ​​nuclease domain comprises the amino acid sequence described in SEQ ID NO:

6.

5. The nuclease according to claim 3, wherein the I-TevI ​​nuclease domain comprises a substitution or deletion of methionine at the first position of SEQ ID NO: 6, and otherwise comprises the amino acid sequence described in SEQ ID NO:

6.

6. The nuclease according to claim 3, further comprising a linker that junctions the modified Staphylococcus aureus Cas9 and the I-TevI ​​nuclease domain.

7. The nuclease according to claim 6, wherein the linker comprises one or more amino acid sequences described in any one of SEQ ID NOs: 7 to 12.

8. The nuclease according to claim 1, bound to a guide RNA.

9. The nuclease according to claim 8, wherein the guide RNA comprises one or more cross-linked nucleic acids.

10. A chimeric nuclease comprising an I-TevI ​​nuclease domain, a linker, Staphylococcus aureus Cas9, and a guide RNA, wherein Staphylococcus aureus Cas9 includes a substitution from aspartic acid to glutamic acid in the amino acid corresponding to the 10th position of SEQ ID NO: 13, and the amino acid sequence of Staphylococcus aureus Cas9 has at least 99% sequence identity with SEQ ID NO:

13.

11. The chimera nuclease according to claim 10, wherein Staphylococcus aureus Cas9 comprises a histidine-to-alanine substitution in the amino acid corresponding to position 557 of SEQ ID NO:

13.

12. The chimeric nuclease according to claim 10, wherein the I-TevI ​​nuclease domain comprises the amino acid sequence described in SEQ ID NO:

6.

13. The chimeric nuclease according to claim 10, wherein the I-TevI ​​nuclease domain comprises a substitution or deletion of methionine at the first position of SEQ ID NO: 6, and otherwise comprises the amino acid sequence described in SEQ ID NO:

6.

14. The chimera nuclease according to claim 10, wherein the linker comprises one or more amino acid sequences described in any one of Sequence IDs 7 to 12.

15. A formulation comprising the chimeric nuclease and lipid nanoparticles according to claim 10.

16. A method for genetically modifying the genome of a cell in vitro or ex vivo, comprising contacting the cell with the chimeric nuclease described in claim 11 in vitro or ex vivo.

17. A nucleic acid encoding a nuclease according to claim 1.

18. A nucleic acid encoding a chimeric nuclease according to claim 10.

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