Nanoparticle compositions for gene therapy

Abstract

A nanoparticle composition comprising a gene editing ribonucleoprotein system complexed in a cationic polymer. The cationic polymer can be a poly-beta amino ester hyperbranched polymer, in particular a 4-branched hyperbranched polymer. The gene editing ribonucleoprotein system can be a CRISPR-Cas9 gene editing system configured to excise a mutation or exon in a gene, replace a mutation in a gene, or create a gene knockdown or knockout, and in particular configured to excise exon 80 of the COL7A1 gene encoding collagen type VII. Data suggests use in.

Description

[0001] This application is a divisional application of application number 2020800666923. Technical Field

[0002] This invention relates to nanoparticle compositions for gene therapy. Methods for treating genetic skin conditions, such as recessive dystrophic epidermolysis bullosa (RDEB), are also envisioned. Background Technology

[0003] Currently, there are no clinical treatments for recessive dystrophic epidermolysis bullosa (RDEB) other than palliative care, making a therapy urgently needed to restore skin structural integrity by inducing the expression of type VII collagen in the patient's own cells. Our research group specializes in designing novel methods to introduce nucleic acids into cells and tissues. Current nucleic acid therapies for restoring type VII collagen expression face safety and efficacy concerns in delivering treatments to cells and tissues. Genome editing, a method of making specific alterations to cellular DNA, can be used to treat conditions like RDEB by repairing disease-causing mutations. Previous genome editing technologies (ZFN and TALEN) have been used to treat RDEB. In recent years, a safer and more versatile new genome editing technology (CRISPR) has attracted considerable interest due to its high therapeutic potential for patients with genetic diseases.

[0004] Therefore, developing safe and efficient delivery systems is crucial for the clinical success of CRISPR genome editing. Despite its enormous potential, overcoming barriers to effective delivery remains critical for achieving safe and effective clinical success. Current methods for delivering CRISPR into cells include: (1) viral vectors; (2) cell electroporation; or (3) polymer vectors. Transient expression of therapeutic Cas9 and guide RNA via non-viral delivery avoids the immune response caused by sustained Cas9 expression and reduces off-target effects in vivo. Safe, non-viral polymer delivery vectors, which can be used in a gentle manner in the CRISPR system approach, correct for type VII collagen in RDEB patients, avoiding the numerous complications and invasive procedures associated with this debilitating condition.

[0005] O'Keefe Ahern et al. (Journal of Investigative Dermatology Vol. 138, No.5. 19 May 2018, pages S141-S141) described the application of CRISPR / Cas9-based COL7A1 genome editing to the treatment of recessive dystrophic epidermolysis bullosa (RDEB) via a non-viral polymer delivery system, specifically highly branched poly(β-amino ester) polymers that bind electrostatically to negatively charged nucleic acids (plasmid DNA).

[0006] Zeng et al. (Nano Letters, Vol. 19, No. 1, 19 December 2018, pages 381-391) describe particles containing DNA and poly(β-amino ester) polymers and their use in fibroblast gene transfection (plasmid DNA).

[0007] Zeng et al. (ACS APPLIED MATERIALS & INTERFACES, Vol. 11, No. 34, 28 August 2019, pages 30661-30672) described nanoparticles containing microcyclic COL7A1 DNA and branched poly(β-amino) esters.

[0008] WO2019 / 104058 describes the delivery of nucleic acids, including ribonucleoproteins, using core-shell structured nanoparticles with a poly(β-amino ester) core encapsulated by a phospholipid bilayer.

[0009] Kang et al. (Bioconjugate Chem 2017, 28, 957-967) described nonviral genome editing using a nanoscale CRISPR complex containing a PEI covalently bound to the Cas9 protein, which is then complexed with a single-guide RNA molecule.

[0010] Chen et al. (ACS APPLIED MATERIALS & INTERFACES, 2018, 10, 18515-18523) described polyplexes formed between nucleic acids (DNA, RNA, or Cas9 / sgRNA ribonucleoproteins) and cationic polymers poly(aspartic acid-(2-aminoethyl disulfide)-(4-imidazolium carboxylic acid))-poly(ethylene glycol).

[0011] Wang et al. (ACS APPLIED MATERIALS & INTERFACES, 2018, 10, 31915-31927) described a polymeric complex formed between nucleic acids (DNA, RNA, or Cas9 / sgRNA ribonucleoprotein) and a cationic copolymer poly(N'N'-bis(acryloyl)cystamine-co-triethylenetetramine).

[0012] The purpose of this invention is to overcome at least one of the problems mentioned above. Summary of the Invention

[0013] The applicant has discovered that hyperbranched poly(β-amino)ester polymers can effectively condense ribonucleoprotein complexes into nanoparticles, protecting them from enzymatic degradation and promoting their efficient and cytocompatible transmembrane transport. It has been demonstrated that the nanoparticle compositions of the present invention, comprising a functional CRISPR-Cas9 type VII collagen exon 80 excision system, can transfect keratinocytes with high transfection efficiency, correction efficiency, and high cell viability. Figures 5 to 7 Two days after local and subcutaneous injection in vivo, these compositions were able to penetrate the basal layer of the epidermis through the blisters of latent dystrophic epidermolysis bullosa (RDEB). Figure 9 After 7 days of topical application of the composition, exon 80 ( ) can be excised in vivo and in vitro. Figure 10 ), and restore type VII collagen in the body ( Figure 11 Compared to CRISPR-plasmid systems, the nanoparticle compositions of this invention exhibit higher transfection efficiency. Figure 12 And higher correction efficiency (8.2% to 43.2%) Figure 13 This invention broadly relates to nanoparticle compositions comprising ribonucleoprotein complexes of hydrophobic cationic polymers and CRISPR-Cas derivatives, and their use in gene therapy (especially for genetic skin conditions), particularly for the treatment of RDEB.

[0014] In a first aspect, the present invention provides a nanoparticle composition comprising a gene editing ribonucleoprotein system compounded in a cationic polymer (e.g., a hyperbranched polymer) (hereinafter referred to as the “nanoparticle composition” or “ribopolymer complex”).

[0015] In one embodiment, the hyperbranched polymer is a poly(β-amino ester) hyperbranched polymer.

[0016] In one embodiment, the hyperbranched polymer is a 3-branched hyperbranched polymer or a 4-branched hyperbranched polymer.

[0017] In one embodiment, the gene-editing ribonucleoprotein system is a Cas9-gRNA ribonucleoprotein system, such as a CRISPR-Cas9 gene-editing system, which is typically configured to induce deletion of a target genomic sequence, including excision of mutations or exons in a gene, replacement of gene mutations, or generation of gene knockdown or knockout. Other gene-editing ribonucleoprotein systems that can be used in this invention include, for example, alternative CRISPR-Cas derivatives, such as Cas12a, Cas14, CRISPR base editors, zinc finger nuclease systems, and TALEN systems. In a preferred embodiment, the gene editing ribonucleoprotein system is the CRISPR-Cas9 gene editing system.

[0018] In one embodiment, the gene-editing ribonucleoprotein system is configured for exon excision. In one embodiment, the gene-editing ribonucleoprotein system is a CRISPR-Cas9 gene-editing system. In one embodiment, the gene-editing ribonucleoprotein system is configured to excise exon 80 of the COL7A1 gene encoding type VII collagen.

[0019] In one embodiment, the average size of the ribopolymer complex of the present invention is 50-500, 50-400, 50-300, 100-400, 100-300, 150-250 nm, and ideally about 200 nm. The average size of the nanoparticle composition is measured, for example, by a dynamic light scattering system or a transmission electron microscope. To measure the size of the ribopolymer complex and to measure the polydispersity index (PDI) of the nanoparticles in solution, a Malvern Zetasizer Nano ZS equipped with a 173° scattering angle can be used. The size measurement of the ribopolymer complex is performed in a transparent disposable plastic cuvette. The ribopolymer complex is prepared as follows: first, sgRNA and Cas9 nuclease are mixed at a desired ratio of 1.1-9.0:1 to assemble a ribonucleoprotein. After assembling the ribonucleoprotein, the polymer and ribonucleoprotein are mixed at a 1:1 volume / volume ratio and incubated at room temperature for 15 minutes to form the ribopolymer complex. After incubation, the ribose polymer complex was further diluted with 980 µl of water and added to a transparent disposable plastic cuvette for measurement at 25°C.

[0020] On the other hand, the present invention provides a composition comprising a first nanoparticle composition according to the invention and a second nanoparticle composition according to the invention, the first nanoparticle composition comprising a first Cas9-gRNA ribonucleoprotein system, and the second nanoparticle composition comprising a second Cas9-gRNA ribonucleoprotein system, wherein the gRNA of the first Cas9-gRNA ribonucleoprotein system is different from the gRNA of the second Cas9-gRNA ribonucleoprotein system. These compositions can be used for exon excision, wherein the first and second gRNA molecules are configured to anneal at opposite flanks of the target exon to be excised. The present invention also provides conjugates comprising the ribopolymer complex according to the invention and other molecules, such as (a) targeting ligands configured to target nanoparticle compositions to specific target cell or tissue types, or (b) imaging markers or dyes. The other molecules may be conjugated to protein or nucleic acid elements of the composition and may be covalently conjugated or otherwise bound, for example, through electrostatic interactions. The present invention also provides pharmaceutical compositions comprising the ribose polymeric complex or conjugate of the present invention and suitable pharmaceutical excipients.

[0021] The present invention also provides a method for preparing a nanoparticle composition, comprising the following steps: Provides a solution for the gene editing ribonucleoprotein system in a buffer solution; Provide a solution of the cationic polymer in a suitable non-aqueous solvent; These solutions were mixed such that the mass of the cationic polymer in the mixture was in excess relative to the mass of the gene-editing ribonucleoprotein system; and The mixture is typically left to stand, allowing it to form a nanoparticle composition.

[0022] In one embodiment, the cationic polymer solution is prepared by dissolving the cationic polymer in a suitable solvent (e.g., a non-aqueous solvent such as DSMO) and then diluting the solution in an aqueous buffer. In one embodiment, the cationic polymer is dissolved in a solvent at a concentration of 10-200 mg / ml, preferably 50-150 mg / ml, and ideally about 100 mg / ml. In one embodiment, the solution diluted with buffer contains 0.1 to 100 g of cationic polymer. In one embodiment, the method includes the step of assembling a gene-editing ribonucleoprotein system. In one embodiment, the step includes mixing sgRNA with Cas9 nuclease at a molar ratio of 1.1-9.0:1 to obtain a gene-editing ribonucleoprotein system typically containing 0.1 to 100 μg of ribonucleoprotein complex. In one embodiment, the ribonucleoprotein complex is diluted in a given volume of buffer solution such that the final ribonucleoprotein complex solution does not exceed 50% of the desired total application volume.

[0023] In one embodiment, the first and second solutions are mixed in a volume ratio of about 1-100:1-100, such that the mass of the polymer is excess relative to the mass of the gene-editing ribonucleoprotein system.

[0024] In one embodiment, the buffer solution is configured to have a pH range of 3 to 10. In one embodiment, the buffer solution contains a buffer salt at a concentration of 10-50 mM, preferably 20-30 mM. In one embodiment, the buffer solution is a sodium acetate buffer. In one embodiment, the content of the cationic polymer in the second solution is 1 to 100 times greater than the content of the ribonucleoprotein complex in terms of mass, wherein the first solution and the second solution are mixed in a volume ratio of about 1-100:1-100.

[0025] The present invention also provides a ribopolymer complex comprising a gene-editing ribonucleoprotein system compounded in a cationic polymer, in a method for treating a genetic disease in an individual (typically characterized by a gene mutation in the individual), wherein in one embodiment, the gene-editing ribonucleoprotein system is configured to edit a gene. Editing may include non-homologous end joining (NHEJ) (i.e., for large or small genomic deletion or exon excision), gene knockdown or knockout, homology-directed repair (HDR), and adding a DNA template to the ribopolymer complex. In a preferred embodiment, editing includes deleting or replacing a mutated or mutated portion of the gene.

[0026] In one embodiment, the ribose polymer complex is administered topically or via subcutaneous injection. In one embodiment, the genetic disease is selected from skin genetic disorders. Examples include epidermolysis bullosa (EB), recessive dystrophic epidermolysis bullosa (RDEB), epidermolysis palmoplantar keratoderma, Hailey-Hailey disease, Darrie's disease, and focal autosomal recessive hypotrichosis. Other skin disorders may include: alternative EB subtypes, such as simple EB and borderline EB, epidermolysis palmoplantar keratoderma, Hailey-Hailey disease, Darrie's disease, and focal autosomal recessive hypotrichosis.

[0027] In one embodiment, the genetic disease is recessive dystrophic epidermolysis bullosa (RDEB), wherein the gene-editing ribonucleoprotein system is configured for exon 80 excision of type VII collagen. Preferably, the gene-editing ribonucleoprotein system is a CRISPR-Cas9 gene-editing system.

[0028] On the other hand, the present invention provides a method for treating a subject's genetic skin disease, comprising the step of applying a nanoparticle composition according to the invention to the individual's skin by topical application or subcutaneous injection, wherein the nanoparticle composition of the invention comprises a gene-editing ribonuclease system typically comprising a CRISPR nuclease complexed with a cationic polymer. Typically, the CRISPR nuclease protein is Cas9 or a Cas9 derivative.

[0029] On the other hand, the present invention provides a method for genetically modifying cells in vitro or in vitro, comprising the step of incubating cells with a nanoparticle composition according to the invention, thereby genetically modifying the cells by the gene-editing ribonucleoprotein system. In one embodiment, the method includes the steps of isolating cells from a subject and then implanting the genetically modified cells into the subject. In one embodiment, the subject suffers from a genetic disease characterized by gene mutations in cells, wherein the gene-editing ribonucleoprotein system is configured to correct or delete the mutation or contain all or part of the exons of the mutation.

[0030] On the other hand, the present invention provides a method for genetically modifying tissue samples in vitro or in vitro, comprising the step of incubating the tissue with a nanoparticle composition according to the invention, thereby genetically modifying at least some cells of the tissue by the gene-editing ribonucleoprotein system. In one embodiment, the method includes the steps of isolating tissue from a subject and then implanting the genetically modified tissue into the subject. In one embodiment, the subject suffers from a genetic disease characterized by gene mutations in tissue cells, wherein the gene-editing ribonucleoprotein system is configured to correct or delete the mutation or contain all or part of the exons of the mutation.

[0031] On the other hand, the present invention provides cells or tissues genetically modified in vitro or ex vivo using the method according to the present invention.

[0032] The present invention also provides a ribopolymer complex comprising a gene-editing ribonucleoprotein system compounded within a cationic polymer for a method of treating an inflammatory disease in an individual characterized by overexpression of inflammatory mediators, wherein the gene-editing ribonucleoprotein system is configured to edit the individual's genome to reduce the expression of inflammatory mediators.

[0033] Other aspects and preferred embodiments of the invention are defined and described in the other claims listed below. Attached Figure Description

[0034] Figure 1 : A schematic diagram defining the ribose polymer complex. Figure 2 : Mechanism of action of ribose polymer complex.

[0035] Figure 3: A schematic diagram of a gene editing strategy using ribose polymer complexes.

[0036] Figure 4 : Formation of ribopolymer complexes used for RDEB treatment.

[0037] Figure 5 Collagen exon 80 excision: The ribonucleoprotein (RNP) complex produces a double-strand break on the flanking side of exon 80, which removes and repairs the DNA strand through non-homologous end joining (NHEJ) and restores the production of type VII collagen.

[0038] Figure 6 RDEBK cell viability 72 hours after transfection with the ribose polymeric complex. RDEB keratinocytes (RDEBK) transfected with the ribose polymeric complex for exon 80 excision of type VII collagen showed high viability compared with untreated cells.

[0039] Figure 7 TracrRNA markers were observed 72 hours after transfection of RDEBK with the ribose polymeric complex, scaled at 100 μm. Fluorescence microscopy images show the fluorescent red markers (tracr) 72 hours after transfection of RDEBK with the 2 μg RNP complex following washing with Hank's solution. Compared to HPAE, the ribose polymeric complex showed the same (P3 polymer), higher (polymers P1 and P2), and more diffuse (Y4 polymer) signal. Figure 7 ).

[0040] Figure 8 Gel electrophoresis images of PCR products transfected with cellular DNA. Gel electrophoresis images of PCR products from primary transfection of keratinocyte DNA demonstrate the correction efficiency of the ribose polymeric complex. Compared to HPAE, using the ribose polymeric complex (Y4 and P2) resulted in shorter bands after exon excision, indicating higher efficiency.

[0041] Figure 9 Fluorescent images of blisters in RDEB human grafts, showing fluorescently labeled red tracrRNA. In an RDEB human transplantation model, after in vivo administration of ribopolymer complexes using different polymers, and two days after local and subcutaneous injections of the ribopolymer complexes, fluorescently labeled red tracrRNA indicates that the ribopolymer complexes penetrated into the epidermal basal layer through RDEB blisters.

[0042] Figure 10 Gel electrophoresis images of DNA amplification (PCR) of humanized RDEB skin grafts treated in vivo and in vitro using a 4-branched polymer gene editing system. In an induced wound model of RDEB human grafts, more than 8% correction was detected 12 days after a single local application in vivo. Figure 10 (Left side). The excised portion of the graft used to create a wound in the animal was immersed in the same ribose polymer complex solution applied to the animal. Samples were analyzed after 6 days of immersion, and exon excision correction reached 33.65% ( Figure 10 (Right side).

[0043] Figure 11 Seven days after local transfection using a 4-branched polymer gene editing system, immunohistochemistry was used to detect type VII collagen fluorescence in human RDEB skin graft samples. This was compared with positive and negative controls for type VII collagen. Figure 11 The comparison on the left side demonstrated that human type VII collagen expression recovered after two treatments with P2 polymer. Figure 11 (Right side). Antibodies with two amino-terminal non-collagenous domains, NC1 (red) and NC2 (green), were used to ensure the function of type VII collagen correction expression. Epidermal protein assays confirmed the human origin of the transplanted skin (red).

[0044] Figure 12 Transfection efficiency of the 3-branched polymer gene editing system: Comparison of plasmid and ribonucleoprotein (RNP). Immortalized human RDEB keratinocytes containing mutant exon 80 were transfected using CRISPR plasmid / 3-branched polymer complex and CRISPR-RNP / 3-branched polymer-ribonucleoprotein complex. The plasmid system (left panel) achieved low transfection efficiency, while the RNP system achieved high transfection efficiency.

[0045] Figure 13 Correction efficiency of the 3-branched polymer gene editing system: Comparison of plasmids and ribonucleoproteins (RNPs): Figure 13 The results show two different PCR amplicon sizes due to the use of different primer systems. At the same w / w ratio, the 3-branched polymer and CRISPR DNA plasmid complex system achieved a correction efficiency of 8.2%, while the CRISPR RNP complex system improved the efficiency to 43.2%. Figure 12 Fluorescence microscopy images and PCR results show that the RNP complex achieved higher transfection and correction efficiency.

[0046] Figure 14 Transfection efficiency of gene-editing ribopolymer complex: Comparison of 3-branched polymer and 4-branched polymer. In porcine primary keratinocytes, the correction efficiency of the Y4 polymer and CRISPR RNP complex system reached 65.98%, significantly higher than the correction efficiency achieved using 3-branched polymer in immortalized cells. Figure 14 Left side). Using Y4 polymers, corrective efficiency can be achieved even in RDEB human primary keratinocytes (cells known to be difficult to transfect). Figure 14(Right side). Detailed Implementation

[0047] All publications, patents, patent applications and other references mentioned herein are incorporated herein by reference in their entirety for all purposes, as if each individual publication, patent or patent application were specifically and individually incorporated herein by reference in its entirety.

[0048] Definitions and general preferences Unless otherwise specified, the following terms as used herein are intended to have the following meanings, in addition to any broader (or narrower) meaning that may have in the art: Unless the context otherwise requires, the singular as used herein shall be understood to include the plural, and vice versa. The terms “a (a)” or “an” used in connection with an entity shall be understood to refer to one or more of that entity. Therefore, the terms “a (a)” (or “an”), “one or more”, and “at least one” are used interchangeably herein.

[0049] As used herein, the term "comprise" or its variations, such as "comprises" or "comprising," should be understood to include any listed whole (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of wholes (e.g., feature, element, characteristic, property, method / process step, or limitation), but does not exclude any other whole or group of wholes. Therefore, as used herein, the term "comprising" is inclusive or open-ended and does not exclude other unlisted wholes or methods / process steps.

[0050] As used herein, the term "disease" is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to include any symptom, disease, abnormality, pathology, unhealthy condition, or syndrome that impairs physiological function, regardless of the nature of the cause (or whether the etiological basis of the disease is actually determined). Therefore, it includes conditions caused by infection, trauma, injury, surgery, radiation ablation, aging, poisoning, or nutritional deficiencies.

[0051] As used herein, the term "treatment" or "treating" refers to an intervention (e.g., administration of a drug to a subject) that cures, improves, or alleviates the symptoms of a disease or eliminates its cause (or reduces its effects) (e.g., reduces the accumulation of pathological levels of lysosomal enzymes). In this context, the term is used synonymously with the term "therapy".

[0052] Furthermore, the terms "treatment" or "treating" refer to an intervention (e.g., administration of a drug to a subject) that prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence in a treated population. In this context, the term "treatment" is used synonymously with the term "prevention."

[0053] As used herein, an effective dose or therapeutically effective dose of a pharmaceutical agent is defined as a dose that can be administered to a subject without excessive toxicity, irritation, allergic reactions, or other problems or complications, is commensurate with a reasonable benefit / risk ratio, and is sufficient to provide the desired effect (e.g., demonstrated by permanent or temporary improvement in the subject's condition to achieve treatment or prevention). Dosage varies from subject to subject, depending on individual age and general condition, route of administration, and other factors. Therefore, while it is impossible to specify an exact effective dose, those skilled in the art will be able to determine an appropriate "effective" dose for any individual using routine experimentation and background common sense. Treatment outcomes described herein include the elimination or reduction of symptoms, reduction of pain or discomfort, prolonged survival, improved mobility, and other markers of clinical improvement. Treatment outcomes are not necessarily a complete cure. Improvements may be observed in terms of biological / molecular markers, clinical, or observational improvements. In preferred embodiments, the methods of the present invention are applicable to humans, large racing animals (horses, camels, dogs), and domestic companion animals (cats and dogs).

[0054] In the context of treatment and effective dosage as defined above, the term "subject" (which, where the context allows, will be understood to include "individual," "animal," "patient," or "mammal") defines any subject to whom treatment is administered, particularly a mammalian subject. Mammal subjects include, but are not limited to, humans, livestock, farm animals, zoo animals, sporting animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cattle, and dairy cows; primates such as apes, monkeys, orangutans, and chimpanzees; canines such as dogs and wolves; felines such as cats, lions, and tigers; equines such as horses, donkeys, zebras, etc.; food animals such as cattle, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters, and guinea pigs. In a preferred embodiment, the subject is a human. As used herein, the term "horse" refers to equine mammals, including horses, donkeys, asses, Tibetan wild asses, and zebras.

[0055] A "gene editing ribonucleoprotein system" or "gene editing RNP system" refers to a complex formed by the binding of a ribosomal protein to one or more nucleic acid sequences, which can edit genes in mammals by, for example, deleting or replacing mutations in genes or gene segments (e.g., exons), inserting oligonucleotides into genes (insertion mutagenesis), or regulating gene expression (knockdown or knockout mutations). The nucleic acid can be RNA, in forms including but not limited to crRNA, TRACNA, and sgRNA. Typically, the nucleic acid is sgRNA, including both crRNA and tracrRNA. The ribosomal protein can be a CRISPR nuclease protein, such as Cas9, Cas12a, Cas14, or Cas variants, such as modified forms of the inactivated nuclease (dCas9). The gene editing ribonucleoprotein system can be further supplemented with nucleic acids added in the form of DNA, RNA, or a combination of both. The supplementary nucleic acid can be integrated into the gene editing ribonucleoprotein system to induce gene enhancement, gene silencing, gene addition, gene knockdown, gene deletion, and gene editing through homology-directed repair. In some embodiments, the nucleic acid can be used in forms including but not limited to RNA oligonucleotides and antisense oligonucleotides. DNA can be used in forms including but not limited to DNA oligonucleotides, antisense oligonucleotides, single-stranded DNA donor oligonucleotides, and plasmid DNA. Gene editing systems can be CRISPR-related Cas systems (Sander and Joung (2014) CRISPR-Cas systems for editing, regulating and targeting genomes Nature Biotechnology 32(4): 347-355), TALEN systems (Boch J (February 2011) "TALEsof genome targeting". Nature Biotechnology. 29(2): 135–6. doi:10.1038 / nbt.1767. PMID 21301438), large-scale nuclease systems, or zinc finger nuclease (ZFN) systems (Carroll, D (2011) "Genome engineering with zinc-finger nucleases". Genetics Society of America. 188(4): 773-78 doi:10.1534 / genetics.111.131433. PMC 3176093). PMID21828278).In one embodiment, the gene editing system is configured to induce insertional mutagenesis in cells, such as the OBLIGARE system and the CRISPR-Cpf1 system (Maresca et al. (2013) Obligate Ligation-Gated Recombination (ObLiGaRe): Custom-designed nuclease-mediated targeted integration through nonhomologous end joining Genome Res. 23: 539-546; see also WO2014 / 033644), Fagerlund et al. (2015) The Cpf1 CRISPR-Cas protein expands genome-editing tools Genome Biology 16:251-253; Ledford (2015) Bacteria yield new gene cutter Smaller CRISPR enzyme should simplify genome editing Nature 526: 17). The gene-editing ribonucleoprotein system of the present invention can be used for gene addition, gene replacement, gene knockdown, and gene editing. Gene replacement is defined as providing a functionally healthy copy of a gene to replace a dysfunctional mutant containing a disease-causing gene. Gene addition is defined as supplementing a therapeutic gene that targets a specific aspect of a disease mechanism. Gene knockdown is defined as the process of inhibiting the ability of a target gene to synthesize toxic / dysfunctional proteins that cause disease. Gene editing is defined as the process of altering the nucleotide sequence of a target gene, thereby causing loss / correction / manipulation of gene expression function. The gene editing systems include, but are not limited to, i) clustered regularly spaced palindromic repeats (CRISPR)-associated (Cas) systems; (ii) transcription activator-like effector nuclease (TALEN) systems; or (iii) zinc finger nuclease (ZFN) systems.

[0056] "Catonic polymers" refer to polymers that carry a positive charge. Examples include LPAE, HPAE, LBPAE, hyperbranched poly-β-amino ester polymers, hyperbranched polymers, hyperbranched poly-β-amino ester polymers, and hyperbranched PEG polymers.

[0057] "Poly-β-amino ester hyperbranched polymer" refers to a cationic polymer formed by random polymerization between branched monomers (e.g., monomers having three, four, or more reaction sites that can react with acrylates or amines), diacrylate groups, and a first amine component and a second amine component, thereby providing a highly branched poly(β-amino ester) (HPAE) with a 3-D structure and multiple end groups. This term includes 3-branched hyperbranched polymers and 4-branched hyperbranched polymers.

[0058] "3-branched hyperbranched polymer" refers to a polymer formed by reacting a monomer (tribranched monomer) having three reaction sites that can react with acrylate or amine groups with a diacrylate, a first amine component, and a second amine component. In one embodiment, the polymer is formed using an oligomer combination method, wherein the diacrylate and the first amine component react together to form a first oligomer, the first oligomer and the second amine component react together to form a second oligomer, and the second oligomer and the tetrabranched monomer react together to form the hyperbranched polymer of the present invention. Zeng et al. (Nano. Lett. 2019 19, 381-391) describe this oligomer combination method in detail. In another embodiment, the tetrabranched monomer, the diacrylate component, and the first amine component react together in a Michael addition reaction to form a first polymer, and the first polymer and the second amine component (terminated amine) react together in a Michael addition reaction to form the hyperbranched polymer of the present invention. Examples of 3-branched hyperbranched polymers are described in US2017216455 and Zeng et al.

[0059] 4-4-branched hyperbranched polymers can be prepared by reacting the following components together: (i) A tetrabranched monomer having four reaction sites that can react with acrylates or amines; (ii) Typically has a diacrylate component of formula (I),

[0060] (I) Z2 is a straight or branched carbon chain with 1 to 30 carbon atoms, a straight or branched carbon chain with 1 to 30 atoms containing heteroatoms, a carbon ring with 3 to 30 carbon atoms, or a heterocycle with 3 to 30 atoms. Z2 is either unsubstituted or substituted with at least one of the following: halogen, hydroxyl, amino, sulfonyl, sulfonamide group, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halogenated C1-C5 alkyl, carboxyl, cyano, nitro, nitroso, -OC(0)NR'R', -N(R')C(0)NR'R, -N(R')C(0)0-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclic, C2-C5 heteroaryl, and C6-C10 aryl; wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl. (iii) Typically contains a first amine component of 3 to 20 atoms. The amine component is generally unsubstituted or substituted with at least one of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halogenated C1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(0)NR'R', -N(R')C(0)NR'R, -N(R')C(0)0-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclic, C2-C5 heteroaryl, and C6-C10 aryl; wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl; and (iv) Typically contains a second amine component of 3 to 20 atoms. The amine component is generally unsubstituted or substituted with at least one of halogen, hydroxyl, amino, sulfonyl, sulfonamide, thiol, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 ether, C1-C6 thioether, C1-C6 sulfone, C1-C6 sulfoxide, C1-C6 primary amide, C1-C6 secondary amide, halogenated C1-C6 alkyl, carboxyl, cyano, nitro, nitroso, -OC(0)NR'R', -N(R')C(0)NR'R, -N(R')C(0)0-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclic, C2-C5 heteroaryl, and C6-C10 aryl; wherein each R' is independently selected from the group consisting of hydrogen and C1-C6 alkyl.

[0061] In one embodiment, a polymer is formed using an oligomer combination method, wherein a diacrylate and a first amine component react together to form a first oligomer, the first oligomer and a second amine component react together to form a second oligomer, and the second oligomer reacts with a tetrabranched monomer to form the hyperbranched polymer of the present invention. Zeng et al. (Nano. Lett. 201919, 381-391) describe this oligomer combination method in detail. In another embodiment, a tetrabranched monomer, a diacrylate component, and a first amine component react together in a Michael addition reaction to form a first polymer, and the first polymer and a second amine component (terminated amine) react together in a Michael addition reaction to form the hyperbranched polymer of the present invention.

[0062] A “tetrabranched monomer” refers to a component having four reactive sites that can react with acrylates or amines. Examples of tetrabranched monomers include diamine and tetraacrylate components, examples of which are provided above. The scaffold can also be a 4-arm PEG component, a pentaerythritol group, a tetraglycidyl group, or a tetrasubstituted silane group. The reactive group can be any acrylamide component (including maleimide), N-hydroxysuccinimide (NHS) component, thiol component, and epoxy component. The following are specific examples of tetrabranched monomers that can be used in the processes and products of this invention: 4-arm PEG acrylamide

[0063] 4-arm PEG-maleimide

[0064] 4-arm PEG-succinimide carbonate NHS

[0065] Tetrathiol component Pentaerythritol tetra(3-mercaptopropionate):

[0066] 4-arm PEG-thiol:

[0067] Tetra(2-mercaptoethyl)silane:

[0068] Tetracyclic oxide component Tetraglycidylmethylenediphenylamine:

[0069] Tetraglycidyl 1,1'-methylenebis(naphthalene-2,7-diol):

[0070] Pentaerythritol tetraglycidyl ether:

[0071] 4-arm polyethylene glycol epoxide

[0072] "Connector" refers to any connector group, including aryl or alkyl. Preferred connectors include O, NH, CH2, alkyl, lower alkyl, alkoxy, lower alkoxy, O-alkyl, CH2O, CH2NH and CH2NHCOCH2, CO, COO.

[0073] "Diamine component" refers to the portion having two functional NH2 groups connected by a linker. "Tetraacrylate" refers to the portion having four acrylate functional groups.

[0074] "Lower alkyl" refers to an alkyl group as defined below, but having 1-10 carbon atoms in its main chain structure, more preferably 1-6 carbon atoms (e.g., "CC-alkyl").

[0075] "Alkyl" refers to a group containing 1 to 8 carbon atoms, which can be straight-chain or branched. An alkyl group is an optionally substituted straight-chain, branched, or cyclic saturated hydrocarbon group. When substituted, an alkyl group can be replaced by no more than four substituents at any available junction. When referring to an alkyl group as alkyl-substituted, this term is used interchangeably with "branched alkyl." Exemplary unsubstituted groups of this type include methyl, ethyl, propyl, isopropyl, α-butyl, isobutyl, pentyl, hexyl, isohexyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, etc. Exemplary substituents may include, but are not limited to, one or more of the following groups: halogens (e.g., F, Cl, Br, I), haloalkyl groups (e.g., CCl3 or CF3), alkoxy groups, alkylthio groups, hydroxyl groups, carboxyl groups (-COOH), alkoxycarbonyl groups (-C(O)R), alkylcarbonyloxy groups (-OCOR), amino groups (-NH2), carbamoyl groups (-NHCOOR- or -OCONHR), urea groups (-NHCONHR-), or thiols (-SH). The defined alkyl groups may also contain one or more carbon double bonds or one or more carbon-carbon triple bonds.

[0076] "Lower alkoxy group" refers to an O-alkyl group, where the alkyl group is as defined above. The alkoxy group is bonded to the core compound via an oxygen bridge. The alkoxy group can be straight-chain or branched; although straight-chain is preferred. Examples include methoxy, ethoxy, propoxy, butoxy, tert-butoxy, isopropoxy, etc. Preferably, the alkoxy group contains 1-4 carbon atoms, particularly preferably 1-3 carbon atoms. The most preferred alkoxy group is methoxy.

[0077] "Halogens" refer to the nonmetallic elements in Group 17 of the periodic table, namely bromine, chlorine, fluorine, iodine, and astatine.

[0078] The terms “alkyl,” “cycloalkyl,” “heterocycloalkyl,” “cycloalkylalkyl,” “aryl,” “acyl,” “aromatic polycycle,” “heteroaryl,” “aranealkyl,” “heteroaryl,” “aminoacyl,” “non-aromatic polycycle,” “mixed aryl and non-aryl polycycle,” “polyheteroaryl,” “non-aromatic polyheterocycle,” “mixed aryl and non-aryl polyheterocycle,” “amino,” and “sulfonyl” are defined in column 4, line 52 to column 7, line 39 of US 6,552,065.

[0079] "Halogens" refer to the nonmetallic elements in Group 17 of the periodic table, namely bromine, chlorine, fluorine, iodine, and astatine.

[0080] "Nanoparticle composition" refers to a composition in the nanoscale range. In one embodiment, the particle composition has a particle size of less than 2 μm, 1.5 μm, 1000 nm, such as 20-900 nm, 50-800 nm, 50-700 nm, 50-600 nm, 50-500 nm, 50-400 nm, 50-300 nm, 100-300 nm, 150-250 nm, or about 200 nm.

[0081] "Gene therapy / editing": This invention can be used to edit a portion of a cell's genome in a targeted and specific manner or to replace a portion of a cell's genome with an exogenous DNA insert.

[0082] Therefore, the present invention can be used to edit or replace defective parts of disease-causing genes (i.e. for gene repair), or to inactivate (i.e. silence) gene insertions that express disease-related genes, or to edit or modify genes, such as deleting disease-causing mutations or modifying or adding residues required for the normal functioning of genes.

[0083] Therefore, the present invention is applied in gene therapy as defined herein.

[0084] The gene therapy according to the present invention can target all cells in an organism, or can target a subpopulation of cells (e.g., a selected organ, tissue or cell).

[0085] The gene therapy according to the present invention can specifically target somatic cells.

[0086] The gene therapy according to the present invention can exclude targeting germ cells. It can exclude targeting totipotent cells. It can exclude targeting human embryos.

[0087] When the gene therapy according to the present invention is applied to selected organs, tissues or cells, the method can be applied ex vivo to isolated organs, tissues or cells (e.g., blood, blood cells, immune cells, bone marrow cells, skin cells, nerve tissue, muscle, etc.).

[0088] Gene therapy can be used to treat any hereditary disease, especially those caused by single-gene mutations. Therefore, gene therapy is particularly used to treat lysosomal storage diseases, muscular dystrophy, cystic fibrosis, Marfan syndrome, sickle cell anemia, dwarfism, phenylketonuria, neurofibromatosis, Huntington's disease, osteogenesis imperfecta, thalassemia, and hemochromatosis.

[0089] Other diseases that may be suitable for gene therapy according to the present invention include: diseases and conditions of the blood, coagulation, heterogeneous skin diseases, cell proliferation and dysregulation, tumor formation (including cancer), inflammatory processes, immune system (including autoimmune diseases), metabolism, liver, kidney, musculoskeletal, nerve, neuronal and eye tissues.

[0090] Exemplary skin conditions include recessive dystrophic epidermolysis bullosa (RDEB), a rare and heterogeneous skin disease caused by a biallelic loss-of-function mutation in the COL7A1 gene. Other skin conditions may include: alternative EB subtypes, such as simple EB and borderline EB, epidermolysis bullosa palmoplantar keratoderma, Hailey-Hailey disease, Darriey's disease, and focal autosomal recessive hypotrichosis.

[0091] Exemplary blood and coagulation disorders and conditions include: anemia, naked lymphocyte syndrome, bleeding disorders, factor H, factor H-like 1, factor V, factor VIII, factor VII, factor X, factor XI, factor XII, factor XIIIA, factor XIIIB deficiency, Fanconi anemia, hemophagocytic lymphocytosis, hemophilia A, hemophilia B, bleeding disorders, leukopenia, sickle cell anemia, and thalassemia.

[0092] Examples of immune-related diseases and conditions include: HIV / AIDS; autoimmune lymphoproliferative syndrome; combined immunodeficiency; HIV-1; HIV susceptibility or infection; immunodeficiency and severe combined immunodeficiency (SCLD). Autoimmune diseases that this invention can treat include Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, vitiligo, type I (early-onset) diabetes, pernicious anemia, multiple sclerosis, glomerulonephritis, systemic lupus erythematosus (SLE, lupus), and Sjögren's syndrome. Other autoimmune diseases include scleroderma, psoriasis, ankylosing spondylitis, myasthenia gravis, pemphigus, polymyositis, dermatomyositis, uveitis, Guillain-Barré syndrome, Crohn's disease, and ulcerative colitis (commonly referred to as inflammatory bowel disease (IBD)).

[0093] Other exemplary diseases include: amyloid neuropathy; amyloidosis; cystic fibrosis; lysosomal storage disease; hepatic adenoma; liver failure; neurological disorders; hepatic lipase deficiency; hepatoblastoma, cancer or carcinoma; medullary cystic kidney disease; phenylketonuria; polycystic kidney disease; or liver disease.

[0094] Exemplary musculoskeletal diseases and conditions include: muscular dystrophy (such as Duchenne and Becker muscular dystrophy), osteoporosis, and muscular atrophy.

[0095] Exemplary neurological and neuronal diseases and conditions include: ALS, Alzheimer's disease; autism; Fragile X syndrome, Huntington's disease, Parkinson's disease, schizophrenia, secretase-related disorders, trinucleotide repeat disorder, Kennedy's disease, Friedrich's ataxia, Machado-Joseph disease, spinocerebellar ataxia, myotonic dystrophy, and dentate nucleus-rubella-lewy body atrophy (DRPLA).

[0096] Exemplary eye diseases include: age-related macular degeneration, corneal opacity and dystrophy, congenital flat keratoplasty, glaucoma, Leber congenital amaurosis, and macular dystrophy.

[0097] The gene therapy according to the present invention is particularly suitable for treating lysosomal storage diseases. Exemplary lysosomal storage diseases and corresponding defective enzymes are listed below: Pompe disease: Acid α-glucosidase Gaucher disease: acid β-glucosidase or glucocerebrosidase Fabry disease: α-galactosidase A GMI-ganglioside storage disease: acid β-galactosidase Tay-Sachs disease: β-hexosaminease A Sankhov disease: β-hexosaminease B Niemann-Pick disease: acid sphingomyelinase Clapham's disease: galactocerebroside lipase Fabry disease: acid ceramide enzyme Metachromatic leukodystrophy: Arylsulfatase A Hurler-Scheie disease: α-L-iduronidase Hunter's disease: Iduuron-2-sulfatase Sanfilippo disease A: Heparin N-sulfatase Sanfilippo disease B: α-N-acetylglucosidase Sanfilippo disease C: Acetyl-CoA: α-aminoglycine N-acetyltransferase Sanfilippo disease D: N-acetylglucosamine-6-sulfatase Morquio disease A: N-acetylgalactosamine-6-sulfatase Morquio disease B: Acid β-galactosidase Maroteaux-Lamy disease: Aromatase B Sly disease: β-glucuronidase Hyper-alpha-mannoside syndrome: Acidic alpha-mannosidase Hyper-β-mannosinolate syndrome: Acid β-mannosidase Fucoside storage disorder: Acidic α-L-fucosidase Sialic acid storage syndrome: sialidase Schindler-Kanzaki disease: α-N-acetylgalactosaminease The gene therapy according to the invention is also particularly suitable for treating proteostatic diseases, including aggregated and misfolded proteostatic diseases such as prions, various amyloidosis and neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease and Huntington's disease), certain forms of diabetes, emphysema, cancer and cystic fibrosis.

[0098] The gene therapy described in this invention is particularly suitable for treating cystic fibrosis. Cystic fibrosis occurs when mutations in the CFTR gene lead to decreased ion channel activity (increased clearance via misfolded CFTR protein).

[0099] The gene therapy described in this invention is particularly suitable for treating diseases caused by amplified CAG repeat sequences. These diseases arise from the amplification of specific gene CAG repeat sequences, in which the encoded proteins have corresponding polyglutamine bundles, leading to aggregation and accumulation in the nucleus and cytoplasm of neurons. The aggregated N-terminal fragments of mutant huntingtin proteins are toxic to neurons and are thought to mediate neurodegeneration. Examples include Huntington's disease (HD), characterized by selective neuronal cell death primarily in the cortex and striatum. CAG amplification has also been found in at least seven other hereditary neurodegenerative diseases, including, for example, spinal and bulbar muscular atrophy (SBMA), Kennedy's disease, certain forms of amyotrophic lateral sclerosis (ALS), dentate nucleus-rubella-lewy body atrophy (DRPLA), and spinocerebellar ataxia types 1, 2, 3, 6, and 7 (SCA).

[0100] The gene therapy according to the present invention is particularly suitable for treating any tumor formation, including proliferative disorders, benign, precancerous and malignant tumor formation, hyperplasia, metaplasia and developmental abnormalities. Therefore, the present invention can be used to treat proliferative disorders, including but not limited to cancer, cancer metastasis, smooth muscle cell proliferation, systemic sclerosis, cirrhosis, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy (e.g., diabetic retinopathy), cardiac hyperplasia, benign prostatic hyperplasia, ovarian cysts, pulmonary fibrosis, endometriosis, fibromatosis, hamartoma, lymphangioma, sarcoidosis and desmoidoma. Tumor formation involving smooth muscle cell proliferation includes excessive proliferation of cells in the vascular system (e.g., endometrial smooth muscle cell proliferation, restenosis and vascular occlusion, especially including stenosis following biological or mechanically mediated vascular injury (e.g., angioplasty)). Furthermore, endometrial smooth muscle cell proliferation can include smooth muscle proliferation outside the vascular system (e.g., obstruction of the bile ducts, bronchial airways and kidneys in patients with renal interstitial fibrosis). Non-cancerous proliferative disorders also include excessive proliferation of skin cells (e.g., psoriasis and its various clinical forms), Reiter's syndrome, pityriasis rubra pilaris, and various proliferative forms of keratosis (including actinic keratosis, senile keratosis, and scleroderma). Treatment of malignant tumor formation (cancer) is particularly preferred.

[0101] application The compositions of the present invention are applicable to topically, orally, rectally, parenterally, intramuscularly, intraperitoneally, intra-arterially, intrabronchially, subcutaneously, intradermally, intravenously, intranasally, vaginally, buccally, ocularly, or sublingually. For oral administration, compressed tablets, pills, tablets, drops, and capsules are particularly used. Preferably, each dose of these compositions contains 0.01 to 250 mg, more preferably 0.1 to 10 mg of the active ingredient. Other forms of administration include solutions or emulsions, which can be injected intravenously, intra-arterially, subcutaneously, intradermally, intraperitoneally, or intramuscularly, and are formulated from sterile or sterile solutions. The pharmaceutical compositions of the present invention can also be in the form of suspensions, emulsions, lotions, ointments, creams, gels, sprays, aerosols, solutions, or powders. The compositions of the present invention can be formulated for topically delivered. Topically delivered generally refers to delivery to the skin, but can also refer to delivery to body cavities lined with epithelial cells, such as the lungs or airways, gastrointestinal tract, or buccal cavity. Specifically, formulations for topical delivery are described in *Topical Drug Delivery Formulations*, edited by David Osborne and Antonio Aman, Taylor & Francis, the entire contents of which are incorporated herein by reference. Compositions or formulations for delivery to the airways are described in papers by O'Riordan et al. (Respir Care, 2002, Nov. 47), EP2050437, WO2005023290, US2010098660, and US20070053845. Compositions and formulations for delivering active agents to the ileum, particularly the proximal ileum, include microparticles and microcapsules, wherein the active agent is encapsulated in a protective matrix formed of polymers or milk proteins, said matrix being acid-resistant but readily soluble in the more alkaline ileal environment. Examples of such delivery systems are described in EP1072600.2 and EP13171757.1. Another transdermal administration method is the use of skin patches. For example, the active ingredient can be incorporated into creams composed of aqueous emulsions of polyethylene glycol or liquid paraffin, or into hydrogels. The active ingredient can also be incorporated at a concentration of 1-10% by weight into ointments composed of a paraffin or nephrite base, along with any necessary stabilizers and preservatives.

[0102] Each injectable dose may contain 10-1000 mg, preferably 10-250 mg of active ingredient.

[0103] The composition can be formulated in unit dose form, that is, in the form of an independent portion containing a unit dose or multiple unit doses or subunit unit doses.

[0104] Those skilled in the art can easily determine the appropriate dosage of the composition of the invention to be administered to a subject without excessive experimentation. Typically, a physician will determine the actual dosage best suited to the individual patient, depending on a variety of factors, including the activity of the particular compound used, the metabolic stability and duration of action of the compound, age, weight, general health condition, sex, diet, method and timing of administration, excretion rate, drug combination, severity of the specific condition, and the individual receiving the therapy. The dosages disclosed herein are examples of average cases. Of course, there may be individual cases requiring higher or lower dosage ranges, which are within the scope of this invention. The dosage of the agent administered is, as needed, from 0.01 to 50 mg / kg body weight, for example, from 0.1 to 10 mg / kg, more preferably from 0.1 to 1 mg / kg body weight.

[0105] The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant, excipient, or carrier administered with the polymeric complex. The drug carrier can be a sterile liquid, such as water and oil, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline solutions, as well as aqueous glucose and glycerol solutions, can also be used as liquid carriers, particularly for injectable liquids. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, limestone, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, or skin penetration enhancers. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc.

[0106] Example The invention will now be described with reference to specific examples. These examples are merely exemplary and for illustrative purposes only; they are not intended to limit the claimed monopoly scope or the described invention in any way. These examples constitute the best mode of practicing the invention as currently contemplated.

[0107] Synthesis of cationic polymer (Y polymer (4-branched diamine)) According to Scheme 1 below, an implementation scheme for preparing 4-branched diamine hyperbranched cationic polymers using BDA, EDA (or HMDA), S5 and DA (or DATA) monomers is described.

[0108] The following monomers were used to synthesize cationic Y polymers: BDA, EDA (or HMDA), S5, and DA (or DATA). Commercially available monomers were copolymerized to form the Y polymer using an “A2+B4+C2” Michael addition strategy. Each selected monomer in the reaction system played a crucial role in the final Y4 polymer. A highly branched polymer was generated by combining a diamine monomer (B4) as the branching unit with a linear diacrylate monomer (A2). Further post-synthetic modification involved end-capping the polymer with other (amine monomer, C2) monomers to remove any unreacted vinyl groups. The monomers were added to a round-bottom flask equipped with a magnetic stirrer. The flask was partially immersed in an oil bath, and polymerization was carried out at 90°C. The progress of the polymer synthesis reaction was monitored by determining the molecular weight, conversion, and PDI using gel permeation chromatography (GPC). When the polymer molecular weight (Mw) approached 10–20 kDa, the reaction was stopped by removing the heat source and diluting with DMSO. Polymer Y chain termination was achieved by reacting the polymer solution with an amine capping agent at room temperature for 48 hours. Following capping, the polymer Y was purified by precipitation twice in excess diethyl ether to remove any remaining residual monomers, unreacted capping agents, and small oligomers. To obtain the final product, the polymer Y was dried in a vacuum oven for 48 hours to remove residual solvents.

[0109] Synthesis of cationic polymers (P polymers (4-branched tetraacrylates, P1, P2, and P3)) According to Scheme 2 below, an implementation scheme for preparing 4-branched tetraacrylate hyperbranched cationic polymers using BDA, PTTA (or DTTA), S5 and DA (or DATA) monomers is described.

[0110] BDA, PTTA (or DTTA), and S5 were mixed in a flask containing DMSO as solvent. The reaction was carried out at 90°C until the target Mw was reached. The reaction was stopped by removing the heat source from the reaction flask and cooling it with ice. The end-capping monomer DA (or DATA) was added to the flask containing DMSO and reacted with the acrylate residue at room temperature for 48 hours. The reaction mixture was then precipitated twice in excess diethyl ether to remove monomers and oligomers. By drying in a vacuum oven, P1 and P2 polymers (BDA+PTTA+S5+DA) with Mws of 7 kDa and 10 kDa, respectively, were obtained. By the same procedure, P3 polymer (BDA+PTTA+S5+DATA) with a Mw of 10 kDa was obtained.

[0111] Synthesis of cationic polymer (HPAE polymer-3-branched triacrylate) 4-Amino-1-butanol (S4), trimethylolpropane triacrylate (TMPTA), and bisphenol A ethoxylated diacrylate (BE) were polymerized via a one-pot “A2+B3+C2” Michael addition reaction. Then, the functional 3-morpholinopropylamine (MPA) was introduced by end-capping to further enhance the properties and function of HPAE as a gene vector (WO2016 / 020474).

[0112]

[0113] Option 1

[0114] Option 2

[0115] Option 3 Synthesis of the nanoparticle composition (ribose polymer complex) of the present invention The ribonucleopolymer complex is formed as follows: Before use, prepare an aqueous solution of the polymer and a solution of the ribonucleoprotein complex in a suitable solvent (e.g., 25 mM sodium acetate). Mix the dissolved ribonucleoprotein and the dissolved polymer together at a 1:1 v / v ratio. Incubate the polymer-ribonucleoprotein complex solution at room temperature for 10 minutes to form the ribonucleopolymer complex. The aqueous solution of the ribonucleopolymer complex should be vigorously mixed to ensure homogeneity. The ribonucleoprotein complex is then compounded with different cationic polymers (P1, P2, and P3 mentioned above).

[0116] For this specific application, a ribonucleoprotein complex was developed for the treatment of recessive dystrophic bullous epidermolysis (RDEB). The strategy for treating RDEB using a ribonucleoprotein complex involves using an RNP complex formed by combining Cas9 and two single-guide RNAs with fluorescently labeled red tracrRNA to exon 80 of type VII collagen. Figure 5 Type VII collagen exon 80 contains numerous RDEB mutations that produce stop codons, such as the most common one being c.6527insC. After exon excision, the resulting type VII collagen fully functions, producing only 30% of the corrective protein required to improve symptoms.

[0117] Both crRNA and tracrRNA were diluted to 100 μM using nuclease-free double buffer, with HiFi Cas9 nuclease used at a stock concentration of 62 μM according to the manufacturer's instructions. RNP complexes were prepared so that the molar ratio of sgRNA (crRNA + tracrRNA):Cas9 was 1–9:1. The premixes were heated in a thermal cycler at 95 °C for 5 minutes to anneal the crRNA and tracrRNA. They were then removed from the heat source and cooled to room temperature on the workbench. HiFi Cas9 nuclease was added to each premix, and the mixtures were allowed to recombine at room temperature in the dark for 15 minutes. Polymer:RNP polymeric complexes (ribose polymeric complexes) were prepared for transfection in a manner similar to that used for plasmid DNA-based transfection. The polymers were diluted to the desired concentration in 25 mM sodium acetate buffer as previously described. Equal volumes of each RNP premix 1 and 2 were used for each transfection. The total weight of the RNP complexes was used to calculate the w / w ratio of the polymers. To form the complex, the polymer solution and RNP solution were mixed at a 1:1 v / v ratio. After mixing by pipetting, the complex was incubated at room temperature for 15 minutes to allow for polymer-RNP interaction. Once incubation was complete, the ribose polymer complex solution was diluted in an appropriate cell culture medium and added to the cells. Four hours after transfection, the medium was changed and replaced with fresh medium. The ATTO 550 nm fluorophore on the tracrRNA was used as an indicator of transfection efficiency.

[0118] viability of transfected cells The alamarBlue™ assay, used to quantitatively determine cell proliferation and metabolic health, was employed to assess cytotoxicity induced by different polymeric complex conditions. Cell viability was assessed 48–72 hours after transfection. Culture medium was removed from cells in each well, and cells were washed with HBSS (Henkes Balanced Salt Solution). Then, 100 μl of alamarBlue™ working solution (10% alamarBlue™ HBSS solution) was added to each well, and the cells were incubated in the dark for 2 hours under normal cell culture conditions. After incubation, the alamarBlue™ solution was transferred to new flat-bottomed 96-well plates, and absorbance at 570 nm and 600 nm was recorded using a SpectraMax M3 multi-plate reader. Readings from wells containing only alamarBlue™ reagent were subtracted from each sample as background readings. Untreated cells were used to normalize fluorescence values ​​and plotted as 100% viability.

[0119] Cell transfection efficiency Cells were seeded 24 to 48 hours prior to transfection to allow for attachment to well plates and flasks. Cells were seeded at an optimized cell density. On the day of transfection, a polymer-DNA complex was prepared and mixed with an appropriate amount of cell culture medium after complexation, ensuring the final polymer-DNA complex solution did not exceed 20% of the total culture medium volume. Cell culture medium containing the polymer-DNA complex was added to the cells, and the medium was removed after 4 hours and replaced with fresh medium to remove the complex.

[0120] In the RDEB human transplantation model, ribopolymer complexes using different polymers were administered in vivo. The in vivo efficiency of the ribopolymer complex can be evaluated in an established humanized mouse model system based on bioengineered human skin grafts from immunodeficient mice, where human fibroblasts and keratinocytes isolated from skin biopsies are expanded in vitro to generate RDEB human bioengineered skin. The tissue bioengineered skin equivalent is then transplanted into athymic mice. This stable implantation of the humanized mouse model represents a useful preclinical platform for modeling pathophysiological processes and testing innovative treatment options. After defining the treatment surface with petrolatum or in a simulated wound within the graft, a suspension of the ribopolymer complex is applied topically and / or intradermally to the RDEB graft. After a certain period, the structural stability of the graft can be tested by mechanically pulling the graft, or the correction efficiency can be assessed by performing a 2mm perforated biopsy. At the end of the evaluation, the graft is analyzed by PCR (…). Figure 10 ), type VII collagen immunofluorescence ( Figure 11 The histological evaluation and the identification of anchored fibrils by transmission electron microscopy (TEM) were used to assess the corrective bands of the transplanted tissue.

[0121] Transfection efficiency of the 3-branched polymer gene editing system: comparison of plasmids and ribonucleoproteins (RNPs) Immortalized RDEB keratinocytes containing the mutant exon 80 were seeded into well plates. After 24 hours, transfection was performed using a CRISPR-Cas9 plasmid or a CRISPR-Cas9-RNP complex to correct the keratinocytes by excising the mutant exon 80 using a dual RNA guide system. The well plates were incubated with the complex for 4 hours, followed by replenishment of medium with fresh medium. Forty-eight hours after transfection, fluorescence images were captured, showing reporter GFP protein (green) and fluorescent tracrRNA (red) markers from the CRISPR-Cas9 plasmid system on the transfected cells. Figure 12 After trypsinizing the cells, DNA was extracted and PCR amplified. The PCR products were run on agarose gel electrophoresis, and the presence of a small band representing the deletion of exon 80 confirmed successful COL7A1 correction. Figure 13Two different PCR amplicon sizes are shown due to the use of different primer systems. At the same w / w ratio, a correction efficiency of 8.2% was achieved using a 3-branched polymer and a CRISPR DNA plasmid, while the efficiency was increased to 43.2% using the CRISPR RNP complex system. Fluorescence microscopy images and PCR results indicate that the RNP complex achieved higher transfection and correction efficiencies.

[0122] Transfection efficiency of gene-editing ribopolymer complexes: Comparison of 3-branched and 4-branched polymers Using the same CRISPR-Cas9 RNP complex and the same protocol as for transfecting immortalized RDEB keratinocytes with 3-branched polymers, primary keratinocytes from different sources (healthy pigs and RDEB humans) were transfected. Notably, 3-branched polymers were used to transfect immortalized cells, which have been shown to be more easily transfected than primary cells. Forty-eight hours after transfection, DNA was extracted and PCR amplified; agarose gel electrophoresis showed corrected bands resulting from exon 80 excision (amplifiers varied in size due to the use of different primers for different cell sources). In porcine primary keratinocytes, the correction efficiency of the Y4 polymer and CRISPR RNP complex system reached 65.98%, significantly higher than the correction efficiency achieved by 3-branched polymers in immortalized cells. Figure 14 Left side). Using Y4 polymers, corrective efficiency can be achieved even in RDEB human primary keratinocytes (cells known to be difficult to transfect). Figure 14 (Right side).

[0123] equivalent The foregoing description has detailed the presently preferred embodiments of the invention. Upon considering these descriptions, those skilled in the art will anticipate that many modifications and variations can be made in their practice. These modifications and variations are all intended to be included in the appended claims.

Claims

1. A nanoparticle composition comprising a gene-editing ribonucleoprotein system composited in a poly-β-amino ester hyperbranched polymer, wherein, The hyperbranched polymer is a 3-branched poly-β-amino ester hyperbranched polymer.

2. The nanoparticle composition according to claim 1, wherein the gene editing ribonucleoprotein system is a CRISPR-Cas gene editing system configured to remove mutations or exons in a gene, replace mutations in a gene, or generate gene knockdown or knockout.

3. The nanoparticle composition according to claim 2, wherein the gene editing ribonucleoprotein system is a CRISPR-Cas9 gene editing system configured to remove exon 80 of the COL7A1 gene encoding type VII collagen.

4. The nanoparticle composition according to any one of claims 1-3, wherein the average size is from 100 nm to 300 nm.

5. A conjugate comprising the nanoparticle composition of any one of claims 1-4, and (a) configured to target the nanoparticle composition to a specific target cell or tissue type by a targeting ligand or (b) an imaging marker.

6. A pharmaceutical composition comprising the nanoparticle composition according to any one of claims 1-4 or the conjugate according to claim 7 and a suitable pharmaceutical excipient.

7. The pharmaceutical composition according to claim 6, wherein it is formulated for topical application to the skin or subcutaneous injection into the skin.

8. A method for preparing a nanoparticle composition, comprising the following steps: Provides a solution for the gene editing ribonucleoprotein system in a buffer solution; Provide a solution of the cationic polymer in a suitable non-aqueous solvent; These solutions were mixed such that the mass of the cationic polymer in the mixture was in excess relative to the mass of the gene-editing ribonucleoprotein system; and The mixture was left to stand, allowing it to form a nanoparticle composition. The hyperbranched polymer is a 3-branched poly-β-amino ester hyperbranched polymer.

9. The method of claim 8, wherein the cationic polymer solution is prepared by dissolving the cationic polymer in DMSO and then diluting the solution in an aqueous buffer.

10. The method of claim 8, wherein the cationic polymer is dissolved in the solvent at a concentration of 10 to 200 mg / mL.

11. The method of claim 9, wherein the cationic polymer solution diluted with buffer contains 0.1 to 100 g of cationic polymer.

12. The method of claim 8, wherein the gene editing ribonucleoprotein system is selected from the group consisting of a CRISPR-Cas gene editing system; a zinc finger nuclease system; and a TALEN system.

13. The method of claim 12, wherein the method comprises the step of assembling a gene-editing ribonucleoprotein system, the step comprising mixing sgRNA with Cas9 nuclease at a molar ratio of 1.1-9.0:1 to obtain a gene-editing ribonucleoprotein system typically containing 0.1 to 100 μg of ribonucleoprotein complex.

14. The method of claim 8, wherein the ribonucleoprotein complex is diluted in a given volume of buffer solution such that the final ribonucleoprotein complex solution does not exceed 50% of the desired total application volume.

15. The method of claim 8, wherein the first solution and the second solution are mixed in a volume ratio of about 1-100:1-100 such that the mass of the cationic polymer is excess relative to the mass of the gene-editing ribonucleoprotein system.

16. The method of claim 8, wherein the buffer solution is configured to have a pH of 3 to 10.

17. The method of claim 8, wherein, in terms of mass, the content of the cationic polymer in the second solution is 1 to 100 times greater than the content of the ribonucleoprotein complex, wherein the first solution and the second solution are mixed in a volume ratio of about 1-100:1-100.

18. Use of the nanoparticle composition of any one of claims 1 to 4 in the preparation of a medicament for treating a genetic disease in an individual, the genetic disease being characterized by a mutation in the individual's gene, wherein the gene-editing ribonucleoprotein system is configured to edit the gene, thereby deleting or replacing the mutated or mutated portion of the gene or restoring the function of the gene.

19. The application according to claim 18, wherein the genetic disease is a skin genetic disease.

20. The application of claim 19, wherein the genetic skin disease is recessive dystrophic epidermolysis bullosa (RDEB), and wherein the gene-editing ribonucleoprotein system is configured for type VII collagen exon 80 skipping.

21. The application according to claim 18, wherein the drug is applied topically or subcutaneously to an individual's skin.

22. The nanoparticle composition according to any one of claims 1 to 4, in an in vitro method for treating a genetic disease in an individual, the genetic disease being characterized by a mutation in a gene in the individual's cells, wherein the gene-editing ribonucleoprotein system is configured to edit the gene to delete or replace the mutated or mutated portion of the gene or to restore the function of the gene, wherein the method includes deleting cells from the individual containing the mutation, treating the cells with the nanoparticle composition to genetically modify the cells, and implanting the genetically modified cells into the individual.

23. The use of the nanoparticle composition of any one of claims 1 to 4 in the preparation of a medicament for treating an individual with an inflammatory disease characterized by overexpression of inflammatory mediators, wherein the gene-editing ribonucleoprotein system is configured to edit the individual's genome to reduce the expression of inflammatory mediators.

24. Use of the nanoparticle composition according to any one of claims 1 to 4 in the preparation of a pharmaceutical product.

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