HYPERBRANCHED CATIONIC POLYMERS USEFUL AS NUCLEIC ACID DELIVERY VECTORS FOR TRANSFECTING CELLS
20220403105 · 2022-12-22
Inventors
- Wenxin WANG (Dublin, IE)
- Sigen A (Dublin, IE)
- Irene Lara-Saez (Valencia, ES)
- Qian Xu (Dublin, IE)
- Jonathan O'Keeffe Ahern (Co. Kildare, IE)
- Dezhong Zhou (Dublin, IE)
Cpc classification
C12N15/87
CHEMISTRY; METALLURGY
C12N15/88
CHEMISTRY; METALLURGY
International classification
C08G83/00
CHEMISTRY; METALLURGY
Abstract
Hyperbranched cationic polymers are described. The polymers employ a 4-branching monomer resulting in an increase in the number of functional terminal groups due to the extra branching units, providing excellent transfection efficiency and cytocompatibility in different cell types, including aADSC, HeLa, Neu7 and RDEB keratinocytes, and delivering different genetic therapy approaches such GFP plasmid DNA and a ribonucleoprotein CRISP-Cas 9 complex for COL7A1 exon 80 skipping. In addition, the extra branching units of the polymer of the invention increases the positive charge on the polymer, which provides for improved endosomal escape within the cell. The 4-branching unit can be a diamine component, or a tetraacrylate component, although other 4-branching monomers may be employed such as for example any component with tetra acrylamide groups (i.e. 4-arm PEG acrylamide, 4-arm PEG maleimide), any component with tetra N- hydroxysuccinimide (NHS) groups (i.e. 4-arm PEG-succinimidyl carbonate NHS ester), any type of tetrathiol component (i.e. Pentaerythritol tetrakis(3-mercaptopropionate), 4-arm PEG-thiol, Tetra(2- mercaptoethyl)silane), and any tetraepoxy component (i.e. TetraGlycidyl methylenedianiline, Tetraglycidyl 1, 1′-methylenebis(naphthalene-2,7-diol), Pentaerythritol tetraglycidyl ether, 4-arm peg epoxide).
Claims
1. A hyperbranched polymer made by reacting together: (i) a four-branching monomer; (ii) a diacrylate component of formula (I) ##STR00019## wherein Z2 is a linear or branched carbon chain of 1 to 30 carbon atoms, a linear or branched heteroatom-containing carbon chains of 1 to 30 atoms, a carbocycle containing 3 to 30 carbon atoms, or a heterocycle containing 3 to 30 atoms; wherein Z2 is unsubstituted or substituted with at least one of a halogen, a hydroxyl, an amino group, a sulfonyl group, a sulphonamide group, a thiol, a C1-C6 alkyl, a C1-C0 alkoxy, a C1-C6 ether, a C1-C6 thioether, a C1-C6 sulfone, a C1-C6 sulfoxide, a C1-C6 primary amide, a C1-C6 secondary amide, a halo C1-C5 alkyl, a carboxyl group, a cyano group, a nitro group, a nitroso group, —OC(0)NR′R′, —N(R′)C(0)NR′R, —N(R′)C(0)0-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 h eteroaryl and C6-C10 aryl; wherein each R′ is independently selected, from the group consisting of hydrogen and C1-C6 alkyl; (iii) a first amine component comprising 3 to 20 atoms, wherein said amine component is unsubstituted or substituted with at least one of a halogen, a hydroxyl, an amino group, a sulfonyl group, a sulphonamide group, a thiol, a C1-C6 alkyl, a C1-C6 alkoxy, a C1-C6 ether, a C1-C6 thioether, a C1-C6 sulfone, a C1-C6 sulfoxide, a C1˜C6 primary amide, a C1-C6 secondary amide, a halo C1-C6 alkyl, a carboxyl group, a cyano group, a nitro group, a nitroso group, —OC(0)NR′R′, - N(R′)C(0)NR′R, —N(R′)C(0)0-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, 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) a second amine component comprising 3 to 20 atoms, wherein said amine component is unsubstituted or substituted with at least one of a halogen, a hydroxyl, an amino group, a sulfonyl group, a sulphonamide group, a thiol, a C1-C6 alkyl, a C1-C6 alkoxy, a C1-C6 ether, a C1-C6 thioether, a C1-C6 sulfone, a C1-C6 sulfoxide, a C1˜C6 primary amide, a C1-C6 secondary amide, a halo C1-C6 alkyl, a carboxyl group, a cyano group, a nitro group, a nitroso group, —OC(0)NR′R′, - N(R′)C(0)NR′R, —N(R′)C(0)0-C1-C6 alkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C5 heteroaryl and C6-C10 aryl; wherein each R′ is independently selected, from the group consisting of hydrogen and C1-C6 alkyl.
2. A hyperbranched polymer according to claim 1, in which the diacrylate and first amine components are reacted together to form a first oligomer, the first oligomer and second amine component are reacted together to form a second oligomer, and the second oligomer and four branching monomer are reacted together to form the hyperbranched polymer.
3. A hyperbranched polymer according to claim 1, in which the four-branching monomer, diacrylate component, and first amine are reacted together in a Michael addition reaction to form a first polymer, and the first polymer and second amine component are reacted together in a Michael addition reaction to form the hyperbranched polymer.
4. A hyperbranched polymer according to claim 1, in which the four-branching monomer is selected from a diamine component, a tetraacrylate component, a tetra acrylamide component, a tetrathiol component, a tetraepoxy component, and a tetra-substituted silane.
5. A hyperbranched polymer according to claim 1, in which the four-branching monomer is selected from the group consisting of: ##STR00020## ##STR00021## ##STR00022##
6. A hyperbranched polymer according to claim 1, in which the diacrylate component of formula (I) is selected from ##STR00023##
7. A hyperbranched polymer according to claim 1, in which the first amine component is selected from: ##STR00024##
8. A hyperbranched polymer according to claim 1, in which the second amine component is a diamine component having a structure NH.sub.2-L.sub.4-NH.sub.2 or is selected from: ##STR00025##
9. A hyperbranched polymer according to claim 1, in which the second amine component is selected from: ##STR00026##
10-20. (canceled)
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0065] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.
Definitions and General Preferences
[0066] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:
[0067] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term “a” or “an” used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.
[0068] As used herein, the term “comprise,” or variations thereof such as “comprises” or “comprising,” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term “comprising” is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.
[0069] 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 encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, age, poisoning or nutritional deficiencies.
[0070] As used herein, the term “treatment” or “treating” refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, the reduction in accumulation of pathological levels of lysosomal enzymes). In this case, the term is used synonymously with the term “therapy”.
[0071] Additionally, the terms “treatment” or “treating” refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term “prophylaxis”.
[0072] As used herein, an effective amount or a therapeutically effective amount of an agent defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate “effective” amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure. Improvement may be observed in biological/molecular markers, clinical or observational improvements. In a preferred embodiment, the methods of the invention are applicable to humans, large racing animals (horses, camels, dogs), and domestic companion animals (cats and dogs).
[0073] In the context of treatment and effective amounts as defined above, the term subject (which is to be read to include “individual”, “animal”, “patient” or “mammal” where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, camels, bison, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human. As used herein, the term “equine” refers to mammals of the family Equidae, which includes horses, donkeys, asses, kiang and zebra.
[0074] “Four branching monomer” refers to a component having a central 4-branching scaffold and four reactive groups capable of reacting acrylate or amine groups. Examples of four- branching monomers include diamine and tetraacrylate components, examples of which are provided above. The scaffold may also be a 4-arm PEG component, a pentaerythritol group, a tetraglycidyl group, or a tetra-substituted silane group. The reactive group may be any acrylamide component (including maleimide), a N-hydroxysuccinimidyl (NHS) component, a thiol component, and an epoxy component. The following are specific examples of four-branching monomers that may be employed in the process and products of the invention:
##STR00010##
Tetrathiol Components
[0075] ##STR00011## ##STR00012##
[0076] “Linker” means any linker group, including an aryl or alkyl group. Preferred linkers include O, NH, CH.sub.2, alkyl, lower alkyl, alkoxy, lower alkoxy, O-alkyl, CH.sub.2O, CH.sub.2NH, and CH.sub.2NHCOCH.sub.2, CO, COO.
[0077] “Diamine component” refers to a moiety having two functional NH2 groups connected by a a linker. “Teraacrylate” refers to a moiety having four functional acrylate groups
[0078] “Lower alkyl” means an alkyl group, as defined below, but having from one to ten carbons, more preferable from one to six carbon atoms (eg. “C—C-alkyl”) in its backbone structure.
[0079] “Alkyl” refers to a group containing from 1 to 8 carbon atoms and may be straight chained or branched. An alkyl group is an optionally substituted straight, branched or cyclic saturated hydrocarbon group. When substituted, alkyl groups may be substituted with up to four substituent groups, at any available point of attachment. When the alkyl group is said to be substituted with an alkyl group, this is used interchangeably with “branched alkyl group”. Exemplary unsubstituted such groups include methyl, ethyl, propyl, isopropyl, a-butyl, isobutyl, pentyl, hexyl, isohexyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. Exemplary substituents may include but are not limited to one or more of the following groups: halo (such as F, Cl, Br, I), Haloalkyl (such as CC13 or CF3), alkoxy, alkylthio, hydroxyl, carboxy (—COOH), alkyloxycarbonyl (—C(O)R), alkylcarbonyloxy (—OCOR), amino (—NH2), carbamoyl (—NHCOOR— or —OCONHR), urea (—NHCONHR—) or thiol (—SH). Alkyl groups as defined may also comprise one or more carbon double bonds or one or more carbon to carbon triple bonds.
[0080] “Lower alkoxy” refers to O-alkyl groups, wherein alkyl is as defined hereinabove. The alkoxy group is bonded to the core compound through the oxygen bridge. The alkoxy group may be straight-chained or branched; although the straight-chain is preferred. Examples include methoxy, ethyloxy, propoxy, butyloxy, t-butyloxy, i-propoxy, and the like. Preferred alkoxy groups contain 1-4 carbon atoms, especially preferred alkoxy groups contain 1-3 carbon atoms. The most preferred alkoxy group is methoxy. “Halogen” means the non-metal elements of Group 17 of the periodic table, namely bromine, chlorine, fluorine, iodine and astatine.
[0081] The terms “alkyl”, “cycloalkyl”, “heterocycloalkyl”, “cycloalkylalkyl”, “aryl”, “acyl”, “aromatic polycycle”, “heteroaryl”, “arylalkyl”, “heteroarylalkyl”, “amino acyl”, “non-aromatic polycycle”, “mixed aryl and non-aryl polycycle”, “polyheteroaryl”, “non-aromatic polyheterocyclic”, “mixed aryl and non-aryl polyheterocycles”, “amino”, and “sulphonyl” are defined in U.S. Pat. No. 6,552,065, Column 4, line 52 to Column 7, line 39.
[0082] “Halogen” means the non-metal elements of Group 17 of the periodic table, namely bromine, chlorine, fluorine, iodine and astatine.
Gene Therapy/Editinq
[0083] The present invention may be used to edit a portion of the genome of a cell or replace a portion of the genome of a cell with an exogenous DNA insert in an orientation-specific manner.
[0084] Thus, the invention may be used to edit or replace a defective portion of a disease-causing gene (i.e. for gene repair), or to insertionally inactivate (i.e. silence) a gene the expression of which is associated with a disease, or to edit or modify a gene for example to delete disease causing mutations or modify or add in residues required for normal functioning of a gene.
[0085] Thus, the invention finds application in gene therapy, as herein defined.
[0086] Gene therapy according to the invention may target all of the cells in an organism, or may be targeted to a subset of cells (e.g. to selected organs, tissues or cells).
[0087] Gene therapy according to the invention may target somatic cells specifically.
[0088] Gene therapy according to the invention may exclude the targeting of germ line cells. It may exclude the targeting of totipotent cells. It may exclude the targeting of human embryos.
[0089] In cases where gene therapy according to the invention is applied to selected organs, tissues or cells, the method may be applied ex vivo to isolated organs, tissues or cells (e.g. to blood, blood cells, immune cells, bone marrow cells, skin cells, nervous tissue, muscle etc.).
[0090] Gene therapy finds application in the treatment of any genetically inherited disorder, particularly those arising from single gene mutations. Thus, gene therapy finds particular application in the treatment of lysosomal storage diseases, muscular dystrophies, cystic fibrosis, Marfan syndrome, sickle cell anaemia, dwarfism, phenylketonuria, neurofibromatosis, Huntington disease, osteogenesis imperfecta, thalassemia and hemochromatosis.
[0091] Other diseases which may be suitable for gene therapy according to the invention include diseases and disorders of: blood, coagulation, heterogenous skin disease, cell proliferation and dysregulation, neoplasia (including cancer), inflammatory processes, immune system (including autoimmune diseases), metabolism, liver, kidney, musculoskeletal, neurological, neuronal and ocular tissues.
[0092] Exemplary skin diseases include recessive dystrophic epidermolysis bullose (RDEB), a rare heterogenous skin disease caused by biallelic loss-of-function mutations in the COL7A1 gene.
[0093] Exemplary blood and coagulation diseases and disorders include: anaemia, bare lymphocyte syndrome, bleeding disorders, deficiencies of factor H, factor H-like 1, factor V, factor VIII, factor VII, factor X, factor XI, factor XII, factor XIIIA, factor XIIIB, Fanconi anaemia, haemophagocytic lymphohistiocytosis, haemophilia A, haemophilia B, haemorrhagic disorder, leukocyte deficiency, sickle cell anaemia and thalassemia.
[0094] Examples of immune related diseases and disorders include: AIDS; autoimmune lymphoproliferative syndrome; combined immunodeficiency; HIV-1; HIV susceptibility or infection; immunodeficiency and severe combined immunodeficiency (SCIDs). Autoimmune diseases which can be treated according to the invention include Grave's disease, rheumatoid arthritis, Hashimoto's thyroiditis, vitiligo, type I (early onset) diabetes, pernicious anaemia, multiple sclerosis, glomerulonephritis, systemic lupus E (SLE, lupus) and Sjogren syndrome. Other autoimmune diseases include scleroderma, psoriasis, ankylosing spondilitis, myasthenia gravis, pemphigus, polymyositis, dermomyositis, uveitis, Guillain-Barre syndrome, Crohn's disease and ulcerative colitis (frequently referred to collectively as inflammatory bowel disease (IBD)).
[0095] Other exemplary diseases include: amyloid neuropathy; amyloidosis; cystic fibrosis; lysosomal storage diseases; hepatic adenoma; hepatic failure; neurologic disorders; hepatic lipase deficiency; hepatoblastoma, cancer or carcinoma; medullary cystic kidney disease; phenylketonuria; polycystic kidney; or hepatic disease.
[0096] Exemplary musculoskeletal diseases and disorders include: muscular dystrophy (e.g. Duchenne and Becker muscular dystrophies), osteoporosis and muscular atrophy.
[0097] Exemplary neurological and neuronal diseases and disorders include: ALS, Alzheimer's disease; autism; fragile X syndrome, Huntington's disease, Parkinson's disease, Schizophrenia, secretase related disorders, trinucleotide repeat disorders, Kennedy's disease, Friedrich's ataxia, Machado-Joseph's disease, spinocerebellar ataxia, myotonic dystrophy and dentatorubral pallidoluysian atrophy (DRPLA).
[0098] Exemplary ocular diseases include: age related macular degeneration, corneal clouding and dystrophy, cornea plana congenital, glaucoma, leber congenital amaurosis and macular dystrophy.
[0099] Gene therapy according to the invention finds particular application in the treatment of lysosomal storage disorders. Listed below are exemplary lysosomal storage disorders and the corresponding defective enzymes: [0100] Pompe disease: Acid alpha-glucosidase [0101] Gaucher disease: Acid beta-glucosidase or glucocerebrosidase [0102] Fabry disease: alpha-Galactosidase A [0103] GMI-gangliosidosis: Acid beta-galactosidase [0104] Tay-Sachs disease: beta-Hexosaminidase A [0105] Sandhoff disease: beta-Hexosaminidase B [0106] Niemann-Pick disease: Acid sphingomyelinase [0107] Krabbe disease: Galactocerebrosidase [0108] Farber disease: Acid ceramidase [0109] Metachromatic leukodystrophy: Arylsulfatase A [0110] Hurler-Scheie disease: alpha-L-Iduronidase [0111] Hunter disease: Iduronate-2-sulfatase [0112] Sanfilippo disease A: Heparan N-sulfatase [0113] Sanfilippo disease B: alpha-N-Acetylglucosaminidase [0114] Sanfilippo disease C: Acetyl-CoA: alpha-glucosaminide N-acetyltransferase [0115] Sanfilippo disease D: N-Acetylglucosamine-6-sulfate sulfatase [0116] Morquio disease A: N-Acetylgalactosamine-6-sulfate sulfatase [0117] Morquio disease B: Acid beta-galactosidase [0118] Maroteaux-Lamy disease: Arylsulfatase B [0119] Sly disease: beta-Glucuronidase [0120] alpha-Mannosidosis: Acid alpha-mannosidase [0121] beta-Mannosidosis: Acid beta-mannosidase [0122] Fucosidosis: Acid alpha-L-fucosidase [0123] Sialidosis: Sialidase [0124] Schindler-Kanzaki disease: alpha-N-acetylgalactosaminidase
[0125] Gene therapy according to the invention also finds particular application in the treatment of proteostatic diseases including both aggregative and misfolding proteostatic diseases, for example prion diseases, various amyloidoses and neurodegenerative disorders (e.g. Parkinson's disease, Alzheimer's disease and Huntington's disease), certain forms of diabetes, emphysema, cancer and cystic fibrosis.
[0126] Gene therapy according to the invention finds particular application in the treatment of cystic fibrosis. Cystic fibrosis occurs when there is a mutation in the CFTR gene leading to reduced ion channel activity (via increased clearance of the misfolded CFTR proteins).
[0127] Gene therapy according to the invention finds particular application in the treatment of expanded CAG repeat diseases. These diseases stem from the expansion of CAG repeats in particular genes with the encoded proteins having corresponding polyglutamine tracts which lead to aggregation and accumulation in the nuclei and cytoplasm of neurons. Aggregated amino-terminal fragments of mutant huntingtin are toxic to neuronal cells and are thought to mediate neurodegeneration. Examples include Huntington's disease (HD), which is characterized by selective neuronal cell death primarily in the cortex and striatum. CAG expansions have also been found in at least seven other inherited neurodegenerative disorders, including for example spinal and bulbar muscular atrophy (SBMA), Kennedy's disease, some forms of amyotrophic lateral sclerosis (ALS), dentatorubral pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia (SCA) types 1, 2, 3, 6 and 7.
[0128] Gene therapy according to the invention finds particular application in the treatment of any neoplasia, including proliferative disorders, benign, pre-cancerous and malignant neoplasia, hyperlasia, metaplasia and dysplasia. The invention therefore finds application in the treatment of proliferative disorders which include, but are not limited to cancer, cancer metastasis, smooth muscle cell proliferation, systemic sclerosis, cirrhosis of the liver, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy (e.g. diabetic retinopathy), cardiac hyperplasia, benign prostatic hyperplasia, ovarian cysts, pulmonary fibrosis, endometriosis, fibromatosis, harmatomas, lymphangiomatosis, sarcoidosis and desmoid tumours. Neoplasia involving smooth muscle cell proliferation include hyperproliferation of cells in the vasculature (e.g. intimal smooth muscle cell hyperplasia, restenosis and vascular occlusion, including in particular stenosis following biologically- or mechanically-mediated vascular injury, such as angioplasty). Moreover, intimal smooth muscle cell hyperplasia can include hyperplasia in smooth muscle other than the vasculature (e.g. blockage of the bile duct, bronchial airways and in the kidneys of patients with renal interstitial fibrosis). Non-cancerous proliferative disorders also include hyperproliferation of cells in the skin such as psoriasis and its varied clinical forms, Reiter's syndrome, pityriasis rubra pilaris and hyperproliferative variants of disorders of keratinization (including actinic keratosis, senile keratosis and scleroderma). Particularly preferred is the treatment of malignant neoplasia (cancer).
[0129] The term “pharmaceutically acceptable excipient” refers to a diluent, adjuvant, excipient, or vehicle with which the polyplex is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
Exemplification
[0130] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.
EXAMPLE 1—Y Polymer (4-branching diamine)
[0131] An embodiment of a 4-branching diamine hyperbranched cationic polymer was prepared according to Scheme 1 below, using BDA, EDA (or HMDA), S5 and DA (or DATA) monomers.
[0132] To synthesize the cationic Y polymer the monomers: BDA, EDA (or HMDA), S5, and DA (or DATA) were used. The Y polymer was formed through an “A2+B4+C2” Michael addition strategy using the copolymerization of commercially available monomers. Each selected monomer within the reaction system plays a pivotal role in the final Y4 polymer. Diamine monomer (B4) was employed as the branching unit to generate the highly branched polymer through combination with linear diacrylate monomer (A2). Further post synthesis modification involved end capping polymer terminal groups with additional (Amine monomer, C2) to remove any unreacted vinyl groups. Monomers were added into a round bottomed flask with a magnetic stirring bar. The flask was placed partially submerged in an oil bath and polymerization reactions were carried out at 90° C. Gel permeation chromatography (GPC) was used to track the progression of the polymer synthesis reaction by measuring molecular weight, conversion and PDI. Upon polymer molecular weight (Mw) approaching 10-20 kDa the reaction was stopped by removing from heat and diluted with DMSO. Y polymers chain termination was achieved by reacting the polymer solution with the amine end capping agent for 48 hrs at room temperature. Post end-capping, Y polymers were purified by precipitating in excess diethyl ether twice so as to remove any remaining residual monomers, unreacted end capping agents and small oligomers. To achieve the final product, Y polymers were dried in a vacuum oven for 48 hrs to remove remaining solvents.
EXAMPLE 2—P Polymer (4-branching tetraacrylate)
[0133] An embodiment of a 4-branching tetraacrylate hyperbranched cationic polymer was prepared according to Scheme 2 below, using BDA, PTTA (or DTTA), S5 and DA (or DATA) monomers. P1 polymer (7 kDa) and P2 polymer (10 kDa). BDA, PTTA (or DTTA), and S5 were mixed into a flask with DMSO as the solvent. The reaction was performed at 90° C. until the target Mw was achieved. The reaction was stopped by removing the reaction flask from heat and cooled with ice. Endcapping monomer DA (or DATA) was added into the flask with DMSO to react with the acrylate residual for 48 hrs at room temperature. Afterwards, the reaction mixture was precipitated into excess amount of diethyl ether twice to remove the monomers and oligomers. The P1 and P2 polymers were achieved by drying in a vacuum oven with 7 kDa and 10 kDa Mw, respectively.
##STR00013## ##STR00014## ##STR00015##
##STR00016##
##STR00017## ##STR00018##
Proton Nuclear Magnetic Resonance (.SUP.1.H-NMR) Measurement
[0134] The chemical structure of Y4 and P polymers was confirmed by .sup.1H-NMR. The polymer was dissolved in deuterated chloroform (CDCl.sub.3) and measurements were carried out on a 300 MHz Bruker NMR equipment. All chemical shifts were reported in ppm relative to tetramethylsilane (TMS).
Molecular Weight Determination by Gel Permeation Chromatography (GPC)
[0135] Molecular weight and polydispersity of the Y4 and P polymers were measured by GPC. A 50 μL of the reaction mixture was collected and diluted into 1 mL DMF, filtered through a 0.22 μm filter and then analyzed using an Agilent 1260 GPC/SEC instrument equipped with a triple detector at 60° C. DMF was used as the mobile phase and the flow rate was 1 mL per min. Poly(methyl methacrylate) was used as the standard sample for GPC calibration.
Synthesis of Polyplexes involving Polymers and Plasmid DNA
[0136] Polyplexes are formed by first preparing aqueous polymer and DNA solutions in a suitable solvent, for example 25 mM sodium acetate, 1:1 v/v ratio of dissolved DNA and dissolved polymer are mixed together, polymer-DNA solution is incubated at room temperature for 10 min to allow polyplex formation prior to use. The aqueous polyplex solution should be mixed vigorously to ensure solution homogeneity.
Cell Viability Studies
[0137] Evaluation of cell cytotoxicity induced by different polyplex conditions has been assessed using the alamarBlue™ assay, which provided a quantitative measurement of cell proliferation and metabolic health. Cell viability has been assessed 48-72 hrs post transfection experiments in cells. Culture media is removed from cells in a well plate and cells are washed with (hanks balanced salt solution) HBSS per well. Following this, 100 μl of alamarBlue™ working solution (10% alamarBlue™ in HBSS) is added to each well and allowed to incubate under normal cell culture conditions for 2 hrs protected from light. After incubation, the alamarBlue™ solution is transferred to a fresh flat bottomed 96 well plate and absorbance at 570 nm and 600 nm is recorded on a SpectraMax M3 multi-plate reader. Wells containing alamarBlue™ reagent only are subtracted from each sample as a background reading. Untreated cells are used to normalize fluorescence values and plotted as 100% viable.
Cell transfection Studies for polyplexes of polymer and DNA
[0138] Cells are seeded 24hr-48hrs prior to transfections to allow attachment to well plates and flasks. Cells are seeded at optimized cell densities. On the day of transfection, polymer-DNA complexes are prepared and after complexation, are mixed with the appropriate cell media such that the final polyplex solution is no more than 20% of the overall media volume. Cell media containing polymer-DNA complexes are added to cells and after 4 hrs is removed and replaced with fresh media to remove complexes.
RNP System
[0139] Both crRNAs and tracrRNA are diluted to 100 μM with nuclease free duplex buffer with HiFi Cas9 nuclease used at stock concentration of 62 μM as per manufacturers guidelines. RNP complexes are prepared such that sgRNA(crRNA+tracrRNA): Cas9 molar ratio is 1-10:1. Master mixes are heated for 5min at 95° C. in a thermocycler to anneal crRNA and tracrRNA. Following this, they are removed from heat and allowed to cool to room temperature on bench top. To each master mix HiFi Cas9 nuclease is added and allowed to complex for 15min at room temperature, protected from light. Polymer:RNP polyplexes (ribopolyplexes) are prepared for transfection in a similar manner to that of plasmid DNA based transfections. Polymer is diluted in 25 mM sodium acetate buffer to desired concentrations as previously described. Equal amounts of each RNP master mix 1 and 2 are used for every transfection. To calculate the w/w ratio for the polymer, the entire weight of the RNP complex is used. To form complexes, polymer solutions are mixed with RNP solutions at a 1:1 v/v ratio. After mixing together by pipetting up and down, complexes are incubated at room temperature for 15 min to allow polymer-RNP interactions. Once incubation is completed, ribopolyplex solutions are ready to be diluted in appropriate cell culture media and added to cells. 4 hrs after transfection, media is changed and replaced with fresh culture media. An ATTO 550 nm fluorophore on the tracrRNA is used an indicator of transfection efficiency.
Equivalents
[0140] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.