Lipid-encapsulated double-strand break endonuclease 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 which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, 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 and lead to a variety of conditions, including early death. Together, they are estimated to affect approximately 6% of people at some point in their lifetime. Diagnosis and treatment for these disorders are often still inadequate, and care is primarily symptomatic, focusing on disease management rather than addressing the underlying genetic defect. There are also many additional disorders in which mutations to genes contribute to the disease pathogenesis.

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

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

[0005] Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CFTR gene, which encodes an epithelial anion channel. The CFTR protein, the cystic fibrosis transmembrane conductance regulator, is found throughout a wide range of organs, including the pancreas, kidneys, liver, lungs, gastrointestinal tract, and reproductive tract, making CF a multisystem disease. Mutations in CFTR result in suboptimal ion transport and fluid retention, leading to the prominent clinical symptoms of abnormal mucus thickening in the lungs and pancreatic insufficiency. In the lungs, dysfunctional CFTR impedes mucociliary clearance, predisposing the organ to bacterial infection and inflammation, ultimately leading to airway obstruction, respiratory failure, and premature death. CF remains the most common and fatal genetic disease in Caucasians, affecting an estimated 70,000 to 100,000 people worldwide, highlighting the real need for the development of better treatments.

[0006] One major challenge to developing therapeutic strategies for CF is the wide variety of mutation types. While the deltaF508 (deletion of phenylalanine at codon 508) mutation is by far the most common in CF patients, with a prevalence of >80%. More than 1,990 deleterious CFTR mutations have been described. These mutations result in premature stop codons, aberrant splicing, incorrect protein folding or transport to the cell surface, and dysfunctional CFTR with limited channel opening. Pharmacological interventions target some of these processes, and while drug administration is therapeutic for some gating mutation types, the commonly occurring deltaF508 mutation still requires more effective treatment. However, pharmaceutical advances in CF care do not address mutations resulting from aberrant splicing or premature stop codons; in these instances, gene editing may prove most advantageous.

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

[0008] Existing gene editing technologies, such as CRISPR-Cas9 (and Cas9 fusions), meganucleases, zinc finger proteins, type II S restriction endonucleases (FokI and FokI fusions), and TALENS, are limited in their ability to introduce specific gene deletions of sufficient length or precisely repair target genes in sufficient cell numbers to be meaningful as therapeutic agents for many genetic diseases. Furthermore, for highly programmable RNA-guided nucleases such as monomeric Cas9, experiments suggest limited specificity for predictably binding, cleaving, and repairing only at their target sites, raising concerns about potential deleterious changes to cellular genomic DNA that could inadvertently cause secondary diseases in patients. Finally, most nucleases are delivered in viral vectors. Viral vectors are subject to the potential for pre-existing immunity in many populations, post-treatment immunogenicity, and genotoxicity. Nonviral delivery methods currently lack the ability to safely deliver nucleases to target cells and enable controlled administration of nucleases in vivo.

[0009] There is an unmet need for improvements to the above-mentioned existing gene editing techniques to address the above concerns, making gene editing techniques more efficient and effective. Summary of the Invention

[0010] The present invention preferably 1 Lacking Lys 26 (The untruncated version of I-TevI ​​contains Lys 27 ) and / or Cys 39 (The untruncated version of I-TevI ​​contains Cys 40 ) modified I-TevI ​​nuclease domain with a linker, in particular SEQ ID NOs: 7 to 12 or fragments thereof, and / or the following mutation Thr 95 (referring to full-length I-TevI), Val 117 , Lys 135 , Gln 158 or Asn 140and may be wild-type or modified versions, preferably containing one or more of the Glu 10 or Ala 557 Chimeric nucleases comprising a modified RNA-guided nuclease Staphylococcus aureus Cas9 containing a mutation, wherein the I-TevI ​​polypeptide comprises the entire amino acid sequence of SEQ ID NO: 6 or a fragment thereof, and a guide RNA targeting the Cas9 domain, particularly SEQ ID NO: 15, 16, 21 or a fragment thereof, and a chimeric nuclease comprising a chimeric nuclease, a cationic and / or neutral lipid nanoparticle, and optionally a DNA-binding compound, particularly GL67 (N 4 -cholesteryl-spermine) and a pharmaceutically acceptable carrier therefor.

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

[0012] Another embodiment of the present invention is directed to a method for editing genes by administering chimeric nucleases to cells or organisms without the use of viral vectors by using controlled administration in vivo.

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

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

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

[0016] Another aspect of the present invention is the use of an extrusion process to produce particles of approximately 100 nM in diameter containing an excipient selected from the group consisting of polysorbate, polyphosphate, calcium chloride, sodium chloride, sodium citrate, sodium hydroxide, sodium phosphate, sodium ethylenediaminetetraacetate, potassium chloride, potassium phosphate and starch or mixtures of these substances so that the novel chimeric nuclease can be administered to a patient using a nebulizer containing the above formulation.

[0017] In a preferred embodiment, the present invention is directed to a method of treating a lung-related disease in a patient in need thereof by administering a novel chimeric nuclease that modifies the DNA of lung epithelial cells, wherein the chimeric nuclease replaces a CFTR delta F508 mutation from the CFTR gene to treat cystic fibrosis or cleaves an 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 is an Ala 10 , Ala 557 or Ala 580 It contains a mutation and targets the EGFR exon 19 deletion in the EGFR gene.

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

[0020] The present invention also encompasses modified donor DNA molecules selected from the group consisting of linkers comprising SEQ ID NOs: 7-12 or fragments thereof, linear single strands of DNA comprising homologous regions adjacent to the site targeted and / or cleaved by the chimeric nuclease, linear double stranded DNA comprising homologous regions adjacent to the site targeted and / or cleaved by the chimeric nuclease, double stranded DNA of the same length comprising complementary DNA ends to that cleaved by the chimeric nuclease, circular double stranded DNA comprising homologous regions adjacent to the site targeted and / or cleaved by the chimeric nuclease, and 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 complementary ends to the ends cleaved by the chimeric nuclease.

[0021] In a further example, a modified GIY-YIG nuclease domain, a linker, and a modified RNA-guided nuclease such as Staphylococcus aureus Cas9 or Streptococcus pyogenes Cas9 or Glu 10 Mutation (SEQ ID NO: 19) and / or Ala 840 The method comprises a chimeric nuclease comprising an EQR Streptococcus pyogenes Cas9 mutant containing a mutation that cleaves the sugar-phosphate backbone of a target DNA on one strand of the target DNA, and the GIY-YIG nuclease domain is selected from the gene family consisting of I-Bmol and Eco29kI.

[0022] In yet further embodiments, the invention includes chimeric nucleases comprising a modified I-TevI ​​nuclease domain, a linker, and a modified nuclease or DNA targeting domain, wherein the modified nuclease or DNA targeting domain is selected from the group consisting of LAGLIDADG, His-Cys Box, HNH, PD-(D / E)xK, and Vsr-like meganucleases, zinc-finger nucleases, scCas9 (Streptococcus canis), fnCas9 (Francisella novicida), cjCas9 (Campylobacter jejuni), Cpf1 (Lachnospiraceae bacterium), Cas12a (Acidaminococcus Sp), and Vsr-like meganucleases. a CRISPR protein selected from the group consisting of Cas13a (Leptorichia shahii), and Cas3 (Streptococcus thermophilus), and a DNA binding domain selected from the group consisting of a zinc-finger motif and a TALE activator domain.

[0023] In yet a further example, the invention encompasses a modified RNA-guided nuclease, Staphylococcus aureus Cas9, and a guide RNA, wherein the guide RNA contains a sequence that targets a genetic polymorphism, a different sequence in the CFTR or EGFR gene, a sequence that retargets the nuclease, a bridging nucleic acid, and / or a mixture of guide RNAs. [Brief explanation of the drawings]

[0024] [Figure 1]Figure 1 is a schematic diagram of the lipid-encapsulated double-cleavage nuclease (TevCas9) after preparation (components not drawn to scale). The I-TevI ​​domain 10 is linked to the RNA-guided dead nuclease (Cas9) domain 12 via a linker domain 11. In a preferred embodiment, the particles formed also contain a guide RNA 13 and donor DNA 14. The nuclease is contained within a lipid particle 15 that is formed into a spherical shape using an extrusion process. [Figure 2] Figure 2 is a diagram of the mechanism by which lipid-encapsulated TevCas9 is internalized into cells and the nucleus to reach its target DNA. As illustrated in Figure 2A, a cell 20 (or cells) is exposed to novel lipid-encapsulated nuclease particles 21 containing TevCas9 25, either by in vivo or ex vivo administration. As shown in Figure 2B, the lipid-encapsulated nuclease particles 21 are endocytosed into the cell 20. They undergo a maturation process in endosomes 22 and are targeted for degradation in the cytoplasm (Figure 2C). In some cases, TevCas9 25 can escape endosomes 22 and enter the cytoplasm (Figure 2D). In eukaryotes, the nuclease (TevCas9) 25 is targeted to the nucleus 23 of the cell 20 via 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 when in the nucleus 23, TevCas9 nuclease 25 binds to and cleaves the target genomic DNA 24 sequence 26. [Figure 3]Figure 3 is a diagram of the mechanism by which lipid-encapsulated TevCas9 modifies target DNA. I-TevI ​​domain 27 targets I-TevI ​​target sequence 29. Linker domain 30 connects I-TevI ​​domain 27 to Cas9 domain 28, which targets Cas9 target sequence 31. Genetic mutation 32 is surrounded by or in close proximity to I-TevI ​​target sequence 29 and Cas9 target sequence 31. As shown in Figure 3B, TevCas9 25 cleaves the target sequence, leaving a deletion product 34 of predictable size with non-complementary DNA ends 35, 36. Figure 3C illustrates that in the presence of single-stranded donor DNA 37 bearing homology arms, cells 20 can insert the donor DNA 37 sequence near the cleavage site via a homology-directed repair (HDR) pathway 38. Figure 3D illustrates that in the presence of donor DNA 39 with compatible DNA ends to those cut by TevCas9 25, the cell 20 can insert the donor DNA sequence 39 between the cuts through directed ligation using the non-homologous end joining (NHEJ) pathway 40. In the absence of donor DNA, the cell 20 can join the DNA ends through the NHEJ pathway 40 (Figure 3E). [Figure 4] Figure 4A demonstrates that TevCas9 targeted to the CFTR gene using the guide in SEQ ID NO: 15 cleaves a CFTR DNA substrate in vitro. Figure 4B shows cells transfected with a plasmid DNA version of TevCas9 fused to a cleavable GFP tag, imaged using phase contrast and GFP imaging on a Cytation 5 (Biotek Instruments Inc, VT, USA) 48 hours after treatment. Genomic DNA is extracted from recovered cells, and editing of the CFTR gene is detected by PCR amplification and a T7 endonuclease I cleavage assay. [Figure 5] Figure 5 shows that TevCas9 targeted to the CFTR delta F508 mutation using the guide in SEQ ID NO: 21 cleaves a DNA substrate containing the CFTR delta F508 mutation in vitro, but does not cleave a substrate containing the wild-type CFTR sequence. [Figure 6] Figure 6A illustrates that the saCas9 D10E mutation slows the conversion of nicked supercoiled DNA to linear DNA. Figure 6B shows that on linear EMX1 DNA substrates, the saCas9 D10E (D10E) ribonucleoprotein complex (RNP) cleaves the target substrate at a level comparable to that of saCas9 wild-type (WT). The computationally predicted level of off-target editing by SaCas9 D10E is lower than that of wild-type saCas9 at the same off-target. [Figure 7] Figure 7A is a schematic diagram of the spacing of I-TevI ​​sites in EGFR exon 19 deletion and wild-type (WT) EGFR. Figures 7B and 7C demonstrate that TevCas9 containing a nicking mutation in Cas9 (H557A) targeted to EGFR using the guide RNA in SEQ ID NO: 16 cleaves EGFR exon 19 deletion DNA substrates at a rate 4-fold faster than wild-type EGFR. Figure 7D is an image showing that HCC827 cells with an EGFR exon 19 deletion mutation treated with TevCas9 targeted to EGFR are selectively killed compared to NuLi-1 cells with wild-type EGFR (WT). DETAILED DESCRIPTION OF THE INVENTION

[0025] Definitions and Acronyms For convenience, certain terms used in the present application, examples, and appended claims are collected here. These definitions should be read in light of the present disclosure and as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0026] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. The term "and / or," as used herein, is defined 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. If a claim recites A and / or B and / or C, the composition may contain 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 of A, B, and C.

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

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

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

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

[0031] The terms "inhalation administration," "inhaling," "inhaled," "inhalation," or "inhalation therapy," which may be used interchangeably and as used herein, include administration of a substantially uniform distribution of appropriately sized particles to the respiratory epithelium of the nose, central airways, peripheral and / or alveolar regions of the lungs, or by intratracheal instillation. Such particles may be introduced into a patient and / or generated using a suitable device, preferably a nebulizer.

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

[0033] The term "pharmaceutically acceptable carrier" is recognized in the art and refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is 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 of being compatible with the subject composition and its components and not harmful to the patient. Some examples of materials that can act as pharmaceutically acceptable carriers include: (1) sugars, such as dextrose, lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as microcrystalline cellulose, sodium carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose (HPMC), and cellulose acetate; (4) glycols, such as propylene glycol; and (5) polyols, such as glycerol, glycerol, sorbitan ... (5) phosphate buffers, such as phosphate buffers, sorbitol, mannitol, and polyethylene glycol; (6) esters, such as ethyl oleate, glyceryl behenate, and ethyl laurate; (7) buffers, such as monobasic and dibasic phosphate, Tris / borate / EDTA, and Tris / acetate / EDTA; (8) pyrogen-free water; (9) isotonic saline; (10) Ringer's solution; (11) ethyl alcohol; (12) phosphate buffers; (13) polysorbates; (14) polyphosphates; and (15) other non-toxic compatible substances used in pharmaceutical formulations. The disclosed excipients can serve multiple functions. For example, solubilizers can 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, refer to an excipient that provides bulk and structure to the lyophilized cake. These crystalline bulking agents are inert and do not react with proteins or nucleic acids. Furthermore, the crystalline bulking agent is capable of crystallizing under lyophilization conditions. Examples of suitable crystalline bulking agents include hydrophilic excipients, such as water-soluble polymers; sugars, such as mannitol, sorbitol, xylitol, glucitol, ducitol, inositol, arabinitol, arabitol, galactitol, iditol, allitol, maltitol, fructose, sorbose, glucose, xylose, trehalose, allose, dextrose, altrose, lactose, glucose, fructose, gulose, idose, galactose, talose, ribose, arabinose, xylose, lyxose, sucrose, maltose, lactose, malt ... Examples of suitable crystalline bulking agents include sucrose, lactulose, fucose, rhamnose, melezitose, maltotriose, raffinose, altritol, their optically active forms (D- or L-forms) and the corresponding racemates; inorganic salts, both mineral and mineral-organic, such as calcium salts, e.g., lactate, gluconate, glyceryl phosphate, citrate, monobasic and dibasic phosphate, succinate, sulfate, and tartrate, as well as aluminum and magnesium salts; carbohydrates, e.g., conventional monosaccharides and disaccharides and the corresponding polyhydric alcohols; proteins, e.g., albumin; amino acids, e.g., 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. Particularly useful bulking agents include dextran.

[0035] The term "pharmaceutically acceptable salts," as used herein, is art-recognized and refers to relatively non-toxic inorganic and organic acid addition salts or inorganic or organic base addition salts of compounds, including, 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 the amino acid X found at the 10th position of a sequence has been replaced with the amino acid Y. As an example, W26C indicates that the amino acid tryptophan-26 (Trp, W) has been changed to a cysteine ​​(Cys). Similarly, the notation AA X indicates that AA is an amino acid that replaces the amino acid found at position X. For example, Lys 26 indicates a substitution of the amino acid at position 26 of the sequence with lysine. The use of either abbreviation is interchangeable. Furthermore, the use of one-letter or three-letter abbreviations for amino acids is also interchangeable.

[0037] The term "therapeutic agent," as used herein, is art-recognized and refers to any chemical or biochemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of therapeutic agents, also referred to as "drugs," are described in well-known sources 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 to treat, prevent, diagnose, cure, or alleviate a disease or disorder; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a physiological environment.

[0038] The term "therapeutic effect," as used herein, is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and especially humans, caused by a pharmacologically active substance. Thus, this term refers to any substance intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease, or in promoting a desired physical or mental manifestation and / or condition in animals or humans. The phrase "therapeutically effective amount" refers to the amount of such a substance that produces some desired local or systemic effect at a theoretical benefit / risk ratio applicable to any treatment. The therapeutically effective amount of such a substance varies depending on the subject and disease state being treated, the subject's weight and age, the severity of the disease state, the mode of administration, etc., and can be readily determined by one of ordinary skill in the art. For example, a composition of the present invention can be administered in an amount sufficient to produce a theoretical benefit / risk ratio applicable to such treatment.

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

[0040] As used herein, "effective amount" refers to an amount sufficient to induce a desired biological response. In the present invention, the desired biological response is treatment of cystic fibrosis and / or non-small cell lung cancer (NSCLC).

[0041] As used herein, a "buffer" refers to any combination of acids or salts that is pharmaceutically acceptable and capable of maintaining the compositions of the present invention within a desired pH range. The buffers in the disclosed compositions maintain a pH in the range of about 2 to about 8.5, about 5.0 to about 8.0, about 6.0 to about 7.5, about 6.5 to about 7.5, or about 6.5. Suitable buffers include any pharmaceutically acceptable buffer capable of maintaining the above pH range, such as acetate, tartaric acid, phosphate, or citrate buffers. In one embodiment, the buffer is a phosphate buffer. In another embodiment, the buffer is an acetate buffer. In one embodiment, the buffer is disodium hydrogen phosphate, sodium chloride, potassium chloride, and potassium phosphate monobasic.

[0042] In the disclosed compositions, the concentration of the buffer solution generally ranges from about 0.1 mM to about 1000 mM, from about 0.2 mM to about 200 mM, from about 0.5 mM to about 50 mM, from about 1 mM to about 10 mM, or about 6.0 mM.

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

[0044] As used herein, an effective amount of antimicrobial agent is an amount effective to inhibit, prevent, or slow the growth of microorganisms, including, for example, bacteria, viruses, and fungi, in the compositions of the present invention. In the compositions of the present invention, the amount of antimicrobial agent is generally in the range of about 0.1 to about 20 mg / ml, about 0.2 to about 30 mg / ml, about 0.2 to about 10 mg / ml, about 0.25 to about 5 mg / ml, about 0.5 to about 50 mg / ml, about 1 to about 10 mg / ml, about 3 mg / ml, or about 5 mg / ml.

[0045] The compositions of the present invention can also be freeze-dried using freeze-drying techniques known in the art and stored as a powder that can be reconstituted before administration.The term "lyophilization" as used herein refers to a freeze-drying or dehydration technique that involves removing a solvent, preferably a water-miscible solvent, more preferably water, from the composition or the present invention, typically by sublimation under high vacuum when the composition is in a frozen state.Generally, freeze-drying is carried out in a freeze-drying device (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, refers to the solid residue or powder produced or remaining after a freeze-drying procedure as described above. The freeze-dried compositions of the present invention generally further comprise a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable excipient," as used herein, refers to a substance added to a solution prior to freeze-drying to enhance characteristics of the freeze-dried cake, such as color, texture, strength, and volume. Pharmaceutically acceptable excipients can be, for example, buffers and pH adjusters, crystal bulking excipients, stabilizers, and tonicity-raising agents.

[0047] As used herein, a stabilizer is a composition that maintains the chemical, biological, or other stability of the 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] Pharmaceutically acceptable salts are salts suitable for administration to a subject, such as a human. The chimeric nucleases of the present invention may have one or more sufficiently acidic protons that can react with a suitable organic or inorganic base to form a base addition salt. Base addition salts include those derived from ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc., and inorganic bases such as organic bases, e.g., alkoxides, alkylamides, alkyl and arylamines. Thus, such bases useful in preparing the salts of the present invention may include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, etc. The chimeric nucleases of the present invention that have a sufficiently basic group, such as an amine, can react with an organic or inorganic acid to form an acid addition salt. Acids commonly used to form acid addition salts from compounds containing basic groups include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of such salts include sulfate, pyrosulfate, bisulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hex ... ,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

[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 review of this specification. The full scope of the present invention, along with such variations, should be determined by reference to the claims, along with their full scope of equivalents and the specification.

[0050] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth, set forth in the specification and claims are understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.

[0051] The above discussion is intended to be illustrative of the principles and various embodiments of the present invention. Numerical variations and modifications will become apparent to those skilled in the art once the above disclosure is fully understood. It is intended that the following claims be interpreted to embrace all such variations and modifications.

[0052] Abbreviation Abbreviations used herein are defined as follows: AA amino acids Cas9 CRISPR-associated protein 9 CF Cystic Fibrosis CFTR Cystic fibrosis transmembrane conductance regulator gene cjCas9 Campylobacter jejuni Cas9 Cpf1 CRISPR from Prevotella and Francisella 1 CRISPR clustered regularly interspaced short palindromic repeats DLS Dynamic Light Scattering DMEM Dulbecco's Modified Eagle's Medium DMPE 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine DNA deoxyribonucleic acid DOAB Dioctadecyldimethylammonium bromide DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DPPC Dipalmitoylphosphatidylcholine E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid EGFR epidermal growth factor receptor ELISA Enzyme-linked immunosorbent assay fnCas9 Francisella novicida Cas9 HDR Homologous Recombination Repair IMAC Immobilized Metal Affinity Chromatography IPTG Isopropyl β-D-1-thiogalactopyranoside MPEG-5000-DMPE N-(carbonyl-methoxypolyethylene glycol 5000)-1,2 dipalmitoyl-sn-glycero-3-phosphoethanolamine NSCLC non-small cell lung cancer NHEJ non-homologous end joining NLS nuclear localization signal PC Phosphatidylcholine PCR polymerase chain reaction PE Phosphoethanolamine RNA ribonucleic acid saCas9 Staphylococcus aureus Cas9 scCas9 Streptococcus canis Cas9 SDS Sodium dodecyl sulfate spCas9 Streptococcus pyogenes Cas9 TALEN transcription activator-like effector nucleases TEV tobacco etch virus TevCas9: A 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 as "TevCas9"), which, when mixed with lipid nanoparticles with or without exogenous donor DNA, upon delivery to cells either replaces DNA sequences in the presence of exogenous donor DNA or deletes defined lengths of DNA in the absence of exogenous donor DNA. The novel chimeric nuclease has been shown to edit genes in human cells as well as cells of other organisms, such as bacteria, yeast, insects, plants, or other mammals, either in whole organisms (in vivo) or in isolated cell culture (ex vivo).

[0054] The novel chimeric nucleases discovered by the inventors exhibit the following advantages over existing gene editing technologies and methods, among others: a. A nuclease that is 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. It can be reprogrammed to many different target DNA sequences through modification of one or more of the I-TevI ​​domain, the linker domain, the Cas9 domain, or the guide RNA (which targets the Cas9 domain to its target sequence). b. If the nuclease cuts at two sites, it excises the correct length of DNA (~30-36 bases depending on the sites targeted by I-TevI ​​and Cas9); c. The Cas9 domain contains a rationally designed mutation (D10E) that alters Cas9 nuclease activity and / or improves the specificity of the Cas9 domain for its target binding site. d. In the presence of exogenous donor DNA, the present invention is designed to replace target DNA sequences in a higher percentage of cells than existing technologies or practices. e. The nuclease can be purified as a single contiguous protein combined with the guide RNA, which simplifies manufacturing; f. Lipid nanoparticles allow for non-viral delivery to target cells with high efficiency and low toxicity, allowing for controlled administration of nucleases. Other lipid-based nuclease delivery technologies exist, but no compositions are suitable for in vivo use; g. Lipid nanoparticles are also designed for delivery of nucleases via nebulization (inhalation); h. One version of the nuclease targets and cleaves the CFTR gene to correct the CFTR delta F508 mutation (SEQ ID NO: 1) for the treatment of cystic fibrosis; i. Another version of the nuclease is designed to target and cleave the clinically relevant EGFR exon 19 deletion mutation (SEQ ID NOs: 2-4), which is present in a variety of cancers, including non-small cell lung cancer (NSCLC).

[0055] Fusion of a GIY-YIG nuclease, such as I-TevI, to a DNA-binding domain via a flexible linker is known (WO 2014 / 121222). A previous version of the dual-cleavage TevCas9 has been described, which includes a linker region comprising amino acids 1-92 of the wild-type I-TevI ​​nuclease domain, 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 nuclease of the present invention includes: i. an I-TevI ​​nuclease domain that binds novel target sequences, allowing targeting of clinically relevant gene sequences, such as the CFTR gene; ii. Various flexible linker regions intended to confer different DNA binding or nuclease activities to TevCas9; iii. saCas9 Nuclease Domain (U.S. Patent Application No. 1988 / 065406B2). The use of saCas9 over spCas9 results in a smaller DNA coding sequence (~3.7 kb for Tev-saCas9 vs. ~4.6 kb for Tev-spCas9) and a lower molecular weight TevCas9 protein (~144 kb for Tev-saCas9 vs. ~179 kb 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 technology, making it more suitable for deletions of defined lengths. iv. A version in which the guide RNA is targeted to a specific CFTR gene sequence near the CFTR delta F508 mutation; and v. A second version in which the guide RNA is targeted to a specific EGFR gene sequence and is intended to cleave only DNA with an appropriate spaced I-TevI ​​site and a Cas9 target site, which are present in certain EGFR exon 19 deletion mutations (SEQ ID NOs: 2-4) but not in wild-type EGFR (SEQ ID NO: 5); a. The present invention includes lipid nanoparticles of certain compositions that are selectively sized to an average diameter of approximately 100 nM. These lipid nanoparticles can deliver nucleases to cells with high efficiency and low toxicity; b. A pharmaceutical formulation of lipids, nucleases, and exogenous donor DNA; c. A pharmaceutical formulation of lipids, nucleases, and exogenous donor DNA that is suitable for nebulization (inhalation); and d. A version of the invention containing exogenous donor DNA that, when delivered with TevCas9 nuclease in a lipid nanoparticle, can integrate into the region between or surrounding two sites targeted by the nuclease.

[0056] The novel chimeric nuclease compositions of the present application contain different combinations of I-TevI ​​domains, linker domains, Cas9 domains, and guide RNAs.

[0057] The versions that target the CFTR gene consist of: i. I-TevI ​​domain of the amino acid sequence according to SEQ ID NO: 6; ii. a linker domain according to any one of SEQ ID NOs: 7 to 12; iii. a saCas9 domain having an amino acid sequence according to SEQ ID NO: 13; and iv. A guide RNA of the RNA sequence according to SEQ ID NO: 15 or 21.

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

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

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

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

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

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

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

[0065] synthesis Example 1: Method for producing TevCas9 nuclease: The DNA coding sequences for the I-TevI ​​domain, linker domain, and Cas9 domain described above were synthesized as a single contiguous DNA sequence using techniques known in the art. Gene synthesis was performed by BioBasic Inc. (Markham, Ontario, 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-kb blocks of the sequence to be synthesized, and these ∼1-kb blocks were synthesized using polymerase chain reaction (PCR). These ∼1-kb blocks were then mixed and subjected to PCR to synthesize the I-TevI ​​domain, linker domain, and Cas9 domain. Furthermore, to facilitate expression of TevCas9 in Escherichia coli (E. coli) and simplify restriction enzyme digestion, the DNA sequence of TevCas9 was optimized prior to synthesis. First, three-base-pair DNA codons used less frequently by Escherichia coli ("E. coli") were replaced with more frequent ones (e.g., of the six codons encoding the amino acid arginine, the relative abundance of the codon AGG was 0.03 compared with 0.42 for the codon CGT). In total, 37% of the codons were changed to those more preferred by 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 an internal sequence. The flanking DNA is digested with the restriction endonucleases NdeI and BamHI (New England Biolabs, Ipswich, MA, United States), in which the target site occurs only once in the DNA sequence, and then inserted using DNA ligase (New England Biolabs, Ipswich, MA, United States) into a similarly digested pET-11a expression vector (EMD Millipore, Burlington, MA, United States), which is suitable for expression of TevCas9 in E. coli.The pET-11a vector containing TevCas9 was transformed into the E. coli expression strain T7 Express (New England Biolabs #C2566, Ipswich, MA, United States), which is optimized for protein expression, including nucleases. Alternatively, the E. coli expression strain BL-21(DE3) (New England Biolabs #C2527, Ipswich, MA, United States) was used. Successful transformation was confirmed by E. coli resistance to ampicillin or tetracycline, and the TevCas9 coding sequence was verified by DNA sequencing of the expression vector from the transformed E. coli. The transformed E. coli was grown at 37°C to an optical density of 0.4-0.6 as measured by a spectrophotometer at a wavelength of 600 nM. Expression of the TevCas9 protein from the pET-11a vector in the transformed E. coli expression strain was induced with IPTG for 10-12 hours at 16°C. Successful expression of TevCas9 was verified by the presence of an approximately 150 kDa band on a Coomassie-stained SDS-polyacrylamide gel in the inducer sample compared to the uninduced sample. E. coli cells were harvested by centrifugation and resuspended in a lysis buffer containing 10 mM imidazole (Sigma, St. Louis, MO, United States), 300–500 mM sodium chloride (Sigma-Aldrich, St. Louis, MO, United States), and 50 mM sodium phosphate (dibasic) (Sigma, St. Louis, MO, United States), pH 8.0 [Buffer 1]. Alternatively, the sodium phosphate (dibasic) in Buffer 1 was replaced with 10 mM Tris-HCl (Sigma, St. Louis, MO, United States), pH 8.0.E. coli is lysed by homogenization using high-pressure liquid homogenizers (Avestin Inc., Ottawa, ON, Canada) or ultrasonicators (Branson Ultrasonics Corp., Danbury, CT, United States) operated at 600-1000 bar, sonication using lysozyme treatment, homogenization using a French pressure cell (Glen Mills Inc., Clifton, NJ, United States), or homogenization using a Dounce homogenizer (Corning Inc., Corning, NY, United States), or any other suitable lysis method known in the art. The lysed material is centrifuged at 12,000 rpm for 20-30 minutes at 4°C, and the supernatant containing soluble TevCas9 is used for subsequent purification steps. The pellet contains cell debris, insoluble intracellular material, and any insoluble TevCas9. Successful lysis and solubility is verified by the presence of an approximately 150 kDa band on a Coomassie-stained SDS-polyacrylamide gel in the supernatant sample compared to the pellet resuspended sample.

[0066] The TevCas9 nuclease is purified in the following steps: 1. The lysate containing the nuclease is loaded onto an immobilized metal affinity chromatography (IMAC) column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) that binds the nuclease. 2. 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-HCl (Sigma, St. Louis, MO, United States), pH 7.6 to 8.0. 4. The eluate is treated with Tobacco Etch Virus (TEV) protease (New England Biolabs, Ipswich, MA, United States) and incubated with the appropriate guide RNA, which is synthesized by Integrated DNA Technology Inc. (Coralville, IA, United States). 5. The treated eluate is loaded back onto the IMAC column and the flow-through containing the TevCas9 nuclease and guide RNA is collected. 6. Successful purification of TevCas9 nuclease is confirmed by the presence of a 150-kilodalton protein band on a Coomassie-stained SDS-polyacrylamide gel. Successful co-purification of TevCas9 with guide RNA is confirmed by treating a sample of the eluate with proteinase K (New England Biolabs, Ipswich, MA, United States), then splitting the sample into two: one subsample is further treated with RNase A (New England Biolabs, Ipswich, MA, United States), and the other is in control buffer without RNase A. A ∼100-nucleotide RNA band is visible on the urea-polyacrylamide gel in the control sample, which is absent in the RNase A-treated sample. 7. Dialyze the solution containing TevCas9 nuclease and guide RNA into a solution containing phosphate buffered saline, pH 7.4.

[0067] Example 2: Method for producing lipid nanoparticles The lipid nanoparticles of the preferred embodiment are composed of one of the following mixtures: I. Lipid nanoparticle 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 fabricated to an average diameter of approximately 100 nM.

[0069] One of lipid mixtures No. 1-3 is selected. For example, DOPE and MPEG-5000-DMPE are mixed together in an appropriate molar ratio in an organic solvent such as chloroform. The organic solvent is then evaporated, and the dried lipid mixture is resuspended by vigorous vortexing in a solution containing phosphate-buffered saline, pH 7.4. The resuspended lipid mixture is then extruded through a 100 nM polycarbonate membrane (T&T Scientific Corporation, Knoxville, TN, United States) equilibrated in phosphate-buffered saline to produce lipid nanoparticles with an average diameter of approximately 100 nM. The solution is filter-sterilized through a 0.2 μM sterile filter (VWR Scientific, Radnor, PA, United States). The mean 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 another suitable technique known in the art.

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

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

[0072] For in vivo cell gene transfer: To assemble lipid-encapsulated TevCas9, lipid nanoparticles are mixed with TevCas9 at a molar ratio of 2000:1 in phosphate-buffered saline and incubated at room temperature for 10 minutes. The molar ratio of lipid-encapsulated TevCas9 / cells for in vivo gene transfer is determined.

[0073] Other embodiments The nucleases may contain different combinations of I-TevI ​​domains, linker domains, Cas9 domains or guide RNAs as highlighted below.

[0074] Modifications 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, Eco29kI, etc. Other versions may also replace the I-TevI ​​domain with other GIY-YIG nuclease domains, such as I-BmoI, Eco29kI, etc. Other versions may also replace the I-TevI ​​domain with MetI as a result of processing when expressed in E. coli. 1 Does not contain.

[0075] Linker domain modifications: The linker domain may include one of the following 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 set forth in SEQ ID NOs: 9-12.

[0076] Modifications of the Cas9 domain: Other versions of the Cas9 domain can include: a) a version of the saCas9 domain containing a D10E mutation (SEQ ID NO: 14); b) a version of the saCas9 domain that nicks 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 but does not cleave the target DNA, e.g., a mutation with both the D10A and H557A mutations (SEQ ID NO: 18); d) a version of the previously described spCas9EQR mutant containing the mutations D1135E, R1335Q, and T1337R combined with the D10E mutation (SEQ ID NO: 19); and e) a version of the previously described spCas9EQR mutant containing the mutations D1135E, R1335Q, and T1337R combined with the D10E mutation and a mutation that nicks 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 can also contain Met 1 Does not contain.

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

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

[0079] Modifications of lipid nanoparticles: a) Other versions of lipid nanoparticles No. 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 peptides that target specific cell types, such as GL67 (N 4 f) the lipid nanoparticles can be lyophilized to promote stability; and g) the lipid nanoparticles can be resuspended in solutions other than phosphate buffered saline, such as sterile isotonic saline, water for injection, etc.

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

[0081] Variations on the method for assembling lipid-encapsulated nuclease and transfecting cells: a) other versions of lipid-encapsulated nuclease may contain different molar ratios of lipid nanoparticles to nuclease; b) media other than DMEM or phosphate-buffered saline may be used for the incubation step; c) the nuclease and lipid nanoparticles may be incubated for less than or more than 10 minutes; and d) other molar amounts of nuclease / cell may be used in the transfection reaction.

[0082] Variations on the method for producing the nuclease: a) other E. coli expression strains can be used, 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); b) buffer 1 or 2 can contain different concentrations of imidazole, sodium chloride, sodium phosphate (dibasic), or tris-HCl and can be buffered to different pHs; c) other processing steps, such as cation or anion exchange chromatography, can be used; d) the nuclease can be dialyzed into solutions other than phosphate-buffered saline, such as sterile isotonic saline, water for injection, etc.; e) the nuclease can be lyophilized to enhance stability; f) guide RNA can be co-expressed from the pACYC-Duet1 expression vector (EMD Millipore, Burlington, MA, United States). g) the DNA coding sequence of the guide RNA is synthesized (Integrated DNA Technology Inc., Coralville, IA, United States), digested with restriction endonucleases, and inserted into a similarly digested second expression site in the pACYC-Duet1 expression vector; g) the guide RNA can be synthesized from double-stranded DNA by transcribing the guide using a T7RNA Polymerase HiScribe Kit (New England Biolabs #E2040S, Ipswich, MA, United States) and purifying the guide using an RNA Cleanup Kit (New England Biolabs #T2030L, Ipswich, MA, United States).

[0083] test Example 1: Methods for demonstrating correction of CFTR delta F508 and CFTR protein function in model cell lines Cultures of 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) in a pharmaceutical formulation targeted to the CFTR delta F508 mutation. An appropriate control cell line, such as NuLi-1 (ATCC® CRL-4011™, American Type Culture Collection, Manassas, VA, United States) immortalized epithelial cells homozygous for wild-type CFTR, is also used.

[0084] The proportion of CFTR delta F508-corrected cells relative to non-corrected 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-amplified target sites (New England Biolabs, Ipswich, MA, United States), deep gene sequencing using an Illumina MiSeq system and barcoded primers flanking the target sites (Illumina, San Diego, CA, United States), or other appropriate methods. The effect of TevCas9 treatment in a control cell line (e.g., NuLi-1 (ATCC® CRL-4011™, American Type Culture Collection, Manassas, VA, United States) will be assessed. CFTR functionality will be measured in treated CuFi-1 cultures (ATCC® CRL-4013™, American Type Culture Collection, Manassas, VA, United States) versus mock-treated CuFi-1 cultures (ATCC® CRL-4013™, American Type Culture Collection, Manassas, VA, United States) using short-circuit current measurements in an Ussing Camber (Warner Instruments, Hamden, CT, United States) in the presence of a chloride gradient. The effect of TevCas9 treatment in a control cell line (e.g., NuLi-1) will also be measured.

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

[0086] The percentage of cells with disrupted EGFR exon 19 deletions relative 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 PCR-amplified target sites (New England Biolabs, Ipswich, MA, United States), deep gene sequencing using the Illumina MiSeq system and barcoded primers flanking the target sites (Illumina, San Diego, CA, United States), or other appropriate methods. EGFR protein expression and activity will also be assessed by TevCas9 treatment in a control cell line (e.g., NuLi-1 (ATCC® CRL-4011™), American Type Culture Collection, Manassas, VA, United States)). EGFR expression is measured using an enzyme-linked immunosorbent assay (ELISA) (Sigma, St. Louis, MO, United States) to detect phosphorylated (i.e., activated), non-phosphorylated, and total EGFR protein in treated HCC827 cultures (ATCC® CRL-2868™, American Type Culture Collection, Manassas, VA, United States) versus mock-treated HCC827 cultures. 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)) is also assessed.

[0087] Example 1: Animal model studies designed to demonstrate efficacy and determine dose-limiting toxicity In an example method demonstrating correction of CFTR delta F508 and / or cystic fibrosis symptoms in animal models (e.g., mice, rats, minipigs, or ferrets) using lipid-encapsulated TevCas9 treatment, lipid-encapsulated TevCas9 targeted to CFTR delta F508 in a pharmaceutical formulation of phosphate-buffered saline, sterile isotonic saline, or water for injection is delivered directly to the lungs by either intubation or intranasal delivery. The procedure time is approximately 30-6000 seconds per treatment, depending on the animal model used.

[0088] Alternatively, lipid-encapsulated TevCas9 in a pharmaceutical formulation targeted to CFTR delta F508 is nebulized using a commercially available nebulizer (Aeroneb®, AeroEclipse®, (Trudell Medical, London, ON, Canada) or PARI-LC Plus®, (PARI USA, Middlesex, VA, United States)). The mean particle size of the lipid nanoparticles, approximately 100 nM, is confirmed post-nebulization by dynamic light scattering (DLS) using a Zetasizer (Malvern Panalytical Ltd, Malvern, United Kingdom) or another suitable technique known in the art. The composition and concentration of lipid-encapsulated TevCas9 are confirmed post-nebulization 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 correction, a representative sheep (minipig) animal model (Exemplar Genetics, Sioux City, IA, United States) homozygous for the CFTR delta F508 mutation is exposed to lipid-encapsulated TevCas9 targeted to CFTR delta F508 and appropriate controls via the oral cavity, nose, or direct lung exposure. General maintenance of these animals includes breeding and farrowing; age-appropriate biosecure housing; sound nutrition; basic vaccinations and veterinary care; and documentation consistent with animal welfare guidelines. Specific maintenance of these animals for 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. Minipigs are treated with a range of lipid-encapsulated TevCas9 concentrations predicted to be effective based on the model cell line experiments described above for 2 days to 4 weeks for acute toxicity studies and up to 24 months for chronic toxicity studies.

[0089] The general health of the animals is monitored after treatment to assess any treatment-related adverse events, such as changes in behavior, weight, or food consumption; immune response; changes to cardiovascular health; mortality, etc. Other measures of efficacy after treatment may include: I. Forced expiratory volume, e.g., forced expiratory volume (or other appropriate method), in each animal after treatment; II. Overall survival of each animal compared to the control; III. Other measures of pulmonary function (e.g., use of mechanical ventilation to allow for general pulmonary function assessment); and IV. Measurement of mutations in vivo through tissue sampling and mutation detection methods, such as by polymerase chain reaction.

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

[0091] The proportion of CFTR delta F508-corrected cells relative to non-corrected 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-amplified target sites (New England Biolabs, Ipswich, MA, United States), deep gene sequencing using an Illumina MiSeq system and barcoded primers flanking the target sites (Illumina, San Diego, CA, United States), or other appropriate methods.

[0092] In a method for demonstrating disruption of EGFR exon 19 deletion mutations and / or non-small cell lung cancer (NSCLC) symptoms with TevCas9 treatment in an animal model, 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 orally, nasally, or directly to the lung. The procedure time is approximately 30-6000 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 nebulized using a commercially available nebulizer (Aeroneb®, AeroEclipse®, (Trudell Medical, London, ON, Canada) or PARI-LC Plus®, (PARI USA, Middlesex, VA, United States)). The mean particle size of the lipid nanoparticles, approximately 100 nM, is confirmed after nebulization by dynamic light scattering (DLS) using a Zetasizer (Malvern Panalytical Ltd, Malvern, United Kingdom) or other suitable techniques known in the art. The composition and concentration of lipid-encapsulated TevCas9 nanoparticles are determined using a MicroGram Lipid Assay Kit (ProFoldin, Hudson, MA, United States). The presence of an approximately 150 kDa band on a Coomassie-stained SDS-polyacrylamide gel is used to confirm the rate of gene disruption after spraying. To measure the rate of gene disruption, a representative murine (mouse) animal model homozygous for the EGFR exon 19 deletion mutation is exposed to lipid-encapsulated TevCas9 targeted to the EGFR exon 19 deletion via the nose, mouth, or direct lung exposure. Mice are treated with various concentrations of TevCas9 predicted to be effective based on 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 general health of the animals is monitored after treatment to assess any treatment-related adverse events, such as changes in behavior, weight, or food consumption; immune response; changes to cardiovascular health; mortality, etc. Other measures of efficacy after treatment may include: I. Quantification of EGFR-activating proteins through positron emission tomography (PET) with EGFR mutant tracers; II. Overall survival of each animal compared to the control; and III. Measurement of tumor formation / remission in each animal over time. In vivo mutation assessment through tissue sampling and mutation detection methods such as the Cobas® EGFR Mutation Test Version 2 (Roche Diagnostics, Risch-Rotkreuz, Switzerland). After treatment with nebulized lipid-encapsulated TevCas9, animals are sacrificed and lung and tracheal tissues are collected.

[0095] The proportion of cells with disrupted EGFR exon 19 deletion mutations relative 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-amplified target sites (New England Biolabs, Ipswich, MA, United States), deep gene sequencing using the Illumina MiSeq system and barcoded primers flanking the target sites (Illumina, San Diego, CA, United States), or other appropriate methods. EGFR protein expression and activity in cells from the collected tissues is measured using an enzyme-linked immunosorbent assay (ELISA) (Sigma, St. Louis, MO, United States) that detects phosphorylated (i.e., activated), non-phosphorylated, and total EGFR protein. To determine dose-limiting toxicity to enable first-in-human clinical trials, various concentrations of lipid-encapsulated TevCas9 (e.g., milligrams per kilogram of body weight) are sprayed and delivered to an appropriate animal model for toxicity testing, such as cynomolgus monkeys or other non-human primates, based on the predicted effective dose from the animal model experiments discussed above. The animals' overall health is monitored for any treatment-related adverse events, such as changes in behavior, weight, or food consumption; immune response; changes to cardiovascular health; and mortality. Other measures of efficacy may be measured in this study, including those described above.

[0096] Treatment effect Although the novel chimeric nucleases of the present invention are intentionally designed to modify the DNA of lung epithelial cells to treat monogenic diseases, 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 a human cell Figure 2 illustrates the mechanism of action of the novel chimeric nuclease cellular uptake of the present invention. As illustrated in Figure 2A, a cell 20 (or cells) is exposed to novel lipid-encapsulated nuclease particles 21 containing TevCas9 25 by either in vivo or ex vivo administration. As shown in Figure 2B, the lipid-encapsulated nuclease particles 21 are endocytosed into the cell 20. Endosomes 22 undergo a maturation process in the cytoplasm and are targeted for degradation (Figure 2C). In some circumstances, TevCas9 25 can escape endosomes 22 and enter the cytoplasm (Figure 2D). In eukaryotes, the nuclease (TevCas9) 25 is targeted to the nucleus 23 of the cell 20 via 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 when in the nucleus 23, TevCas9 nuclease 25 binds to and cleaves the target genomic DNA 24 sequence 26.

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

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

[0100] Example 3: Treatment of non-small cell lung cancer For applications to treat non-small cell lung cancer, we used a version of the Cas9 domain (D10A or H557A mutation) or a nuclease-deficient version (D10A+H557A mutation) that cleaves only one strand of DNA, 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 the two sites targeted by the nuclease by non-homologous end joining (Figure 3E).

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

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

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

[0104] Nebulizers may require several steps to prepare the liquid for nebulization. The medication is typically held in liquid form in a cup inside the nebulizer chamber. Once loaded, the device is turned on, generating compressed air to convert the liquid into a vapor in the nebulizer chamber. The patient places the mouthpiece of the nebulizer chamber over their mouth, takes a sharp, deep inhalation, and holds their breath for 5 to 10 seconds to ensure the medication reaches the lower lungs. A variety of such devices exist. Many modern nebulizers are breath-activated and rely on the patient's inhalation force to synchronize the aerosolized liquid from the device, thus ensuring that the medication is delivered only to the patient and not to the surrounding environment. This also ensures consistency in the delivery of the entire dose of medication to the patient.

[0105] The use of nebulizers is well known, and nebulizers are commercially available from several sources, such as Aeroneb®, AeroEclipse® (Trudell Medical, London, ON, Canada), or PARI-LC Plus® (PARI USA, Middlesex, VA, United States). In an embodiment of the present invention, a nebulizer is utilized for delivery of the present lipid-encapsulated novel chimeric nuclease comprising a modified I-TevI ​​nuclease domain, a linker, and a modified RNA-guided nuclease, Staphylococcus aureus Cas9, to lung epithelial tissue. A sterile liquid version of the therapeutic agent of interest is placed into the nebulizing chamber, subsequently aerosolized, and inhaled by the patient into the lungs via a deep breath.

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

[0107] No non-invasive delivery route offers the speed of action that inhaled drugs can provide. One advantage of inhaled drugs is that they are absorbed more rapidly than subcutaneously injected molecules, providing a more immediate physiological response. Small or large molecules, especially hydrophobic molecules, can be absorbed within seconds after inhalation and therefore can be used to treat a wide variety of symptoms that occur suddenly or require long-term administration. Pain, panic, anxiety, nausea, cardiovascular crisis, 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 have some water solubility and are rapidly and effectively absorbed from the lungs. Those with higher hydrophobicity are absorbed even more rapidly, within seconds to minutes. Those with higher hydrophilicity are absorbed within minutes to 10 minutes. In one example of the present invention, for pulmonary delivery of a therapeutic dose, one vial is aseptically filled with a therapeutic dose of hydrophobic lipid nanoparticles, and another vial is aseptically filled with a therapeutic dose of a chimeric nuclease comprising a water-soluble and hydrophilic modified I-TevI ​​nuclease domain, a linker, and a modified RNA-guided nuclease, Staphylococcus aureus Cas9. The dose can range from 1 to 1000 milligrams of each lipid nanoparticle and chimeric nuclease, with approximately 5 to 200 milligrams being preferred. The claimed lipid-encapsulated chimeric nuclease, containing a modified I-TevI ​​nuclease domain, a linker, and the modified RNA-guided nuclease Staphylococcus aureus Cas9, can be absorbed by lung cells within hours, and complete cleavage of DNA substrates in vitro was observed within two hours. Other nebulized therapeutics have been delivered daily. Nebulized administration of the lipid-encapsulated chimeric nuclease can therefore be daily or less frequent, depending on its efficacy in patients. 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 varies depending on the condition, age, and weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the subject composition. Any of the subject formulations may be administered in a single dose or in divided doses. The dosage for the present compositions can be easily determined by techniques known to those skilled in the art or as taught herein.

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

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

[0112] The exact time and amount of administration of any particular subject composition that will result in the most effective treatment in a given patient will depend on the activity, pharmacokinetics, and bioavailability of the subject composition, the physiological condition of the patient (including age, sex, disease type and stage, general health, response to a given dosage and type of drug), route of administration, etc. The guidance provided herein can be used to optimize treatment, e.g., by determining optimal time and / or dosage, which will require no more than routine experimentation consisting of monitoring subjects and adjusting dosage and / or timing.

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

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

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

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

[0117] The data obtained from cell culture assays and animal studies may be used in formulating a range of dosage for human use. The dosage of any subject composition is preferably within the ED range with little or no toxicity. 50 The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. For compositions of the present disclosure, the therapeutically effective dose can be estimated initially from cell culture assays.

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

[0119] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of a composition that may be combined with a carrier material to produce a single dosage will vary depending upon the subject being treated and the particular mode of administration.

[0120] Methods of preparing these formulations include the step of bringing into association compositions of the present disclosure with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the agent with the liquid carrier.

[0121] It will be appreciated that the disclosed compositions can include lyophilized or freeze-dried compounds disclosed herein. For example, compositions are disclosed herein that are crystalline and / or amorphous powder forms of the disclosed compounds. Such forms can be reconstituted 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 subject composition, 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, tetrahydrofuryl alcohol, and fatty acid esters of sorbitan, cyclodextrin, albumin, hyaluronic acid, chitosan, and mixtures thereof. Polyethylene glycol (PEG) may be used to achieve desired properties of solubility, stability, half-life, and other pharmaceutically beneficial properties. Representative examples of stabilizing ingredients 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, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinol 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] Usually, aqueous aerosols are prepared by formulating aqueous solutions or suspensions of the subject compositions together with conventional pharmaceutically acceptable carriers and stabilizers.Carriers and stabilizers vary according to the requirements of specific subject compositions, but generally include nonionic surfactants (Tweens, pluronics or polyethylene glycol), innocuous proteins such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.Aerosols are generally prepared from isotonic solutions.

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

[0125] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (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. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0126] The compositions of the present invention are generally sterile, storage-stable, and pharmaceutically acceptable aqueous solutions that can be easily administered without the need for reconstitution before administration. The compositions of the present invention are suitable for administration to a subject, meaning that they are pharmaceutically acceptable, non-toxic, do not contain any components that adversely affect the biological effect of the chimeric nuclease, and have a pH close to that of physiological conditions to avoid reactions at the inhalation and / or injection site. The compositions of the present invention do not contain, for example, cells.

[0127] The 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 the intrusion of external components, such as microorganisms, when maintained at 25°C for at least 3 months.

[0128] The compositions of the present invention are preferably administered by nebulization, typically by breath-actuated nebulization.

[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 additional therapeutic agents, such as antiviral agents, antimicrobial agents, chemotherapeutic agents and immunotherapeutics.

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

[0132] As discussed above, the compositions of the present invention can be administered by nebulization. Suitable volumes of the compositions of the present invention for nebulization 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 the chimeric nuclease are about 0.1 mg / ml to about 10.0 mg / ml, about 10.0 mg / ml to about 100.0 mg / ml, about 30.0 mg / ml to about 300.0 mg / ml, about 500 mg / ml to about 2000 mg / ml, and about 2.0 mg / ml.

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

Claims

[Request 1] 14 or a fragment thereof, provided that the fragment is Glu. １０ A polypeptide containing a mutation.

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