Nucleic acid products and methods of administration thereof
The present invention relates in part to nucleic acids, including nucleic acids encoding proteins, therapeutics and cosmetics comprising nucleic acids, methods for delivering nucleic acids to cells, tissues, organs, and patients, methods for inducing cells to express proteins using nucleic acids, methods, kits and devices for transfecting, gene editing, and reprogramming cells, and cells, organisms, therapeutics, and cosmetics produced using these methods, kits, and devices.
1. A method for gene-editing a cell in the skin of a human subject, comprising inducing a single-strand or double-strand break in the DNA of the cell, comprising:
administering intradermally to the human subject a composition comprising an effective dose of about 300 ng of an RNA encoding a protein of interest to result in the cell expressing the protein of interest,
wherein the protein of interest is a gene-editing protein, and
wherein the effective dose of about 300 ng of the RNA encoding the protein of interest provides greater expression of the protein of interest than a dose of 1200 ng of RNA encoding the protein of interest during the second to fifth day after administering the RNA.
2. The method of claim 1 , wherein the RNA comprises one or more of 5-hydroxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, 5-methoxypseudouridine, and 7-deazaguanosine.
3. The method of claim 1 , wherein the RNA comprises a 5′ cap structure, a 5′-UTR comprising a Kozak consensus sequence, a 5′-UTR comprising a sequence that increases RNA stability in vivo, a 3′-UTR comprising a sequence that increases RNA stability in vivo, and/or a 3′ poly(A) tail.
4. The method of claim 1 , wherein the RNA is from about 200 nucleotides to about 5000 nucleotides in length, about 500 to about 2000 nucleotides in length, about 500 to about 1500 nucleotides in length, or about 500 to about 1000 nucleotides in length and the RNA is prepared by in vitro transcription.
5. The method of claim 1 , wherein the composition comprises one or more lipids that enhance uptake of RNA by cells.
6. The method of claim 5 , wherein the one or more lipids are selected from 3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol; 1,2-dioleoyl-3-trimethylammonium-propane; N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium; 1,2-dimyristoyl-3-trimethylammonium-propane; 1,2-dipalmitoyl-3-trimethylammonium-propane; 1,2-stearoyl-3-trimethylammonium-propane; 1,2-dioleoyl-3-dimethylammonium-propane; 1,2-dimyristoyl-3-dimethylammonium-propane; 1,2-dipalmitoyl-3-dimethylammonium-propane; 1,2-distearoyl-3-dimethylammonium-propane; dimethyldioctadecylammonium; 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine; 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine; 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine; 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine; 1,2-distearoyl-sn-glycero-3-ethylphosphocholine; 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine; 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine; 1,2-di-O-octadecenyl-3-trimethylammonium propane; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide; 2,3-dioleyloxy-N-[2-spermine carboxamide]ethyl-N,N-dimethyl-1-propanammonium trifluoroacetate; 1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid; N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide; a transfection reagent; dioctadecyl amidoglyceryl spermine; dioleoyl phosphatidyl ethanolamine; and combinations thereof.
7. The method of claim 1 , wherein about 300 ng of RNA is administered per treatment area of about 10 cm 2 or less, or about 5 cm 2 or less, or about 1 cm 2 or less, or about 0.5 cm 2 or less, or about 0.2 cm 2 or less.
8. The method of claim 1 , wherein the composition is administered about weekly for at least 2 weeks, is administered about every other week for at least one month, is administered monthly, or is administered about every other month.
9. The method of claim 8 , wherein the composition is administered for at least two months, or at least 4 months, or at least 6 months, or at least 9 months, or at least one year.
10. The method of claim 1 , wherein the human subject has a disease, disorder, or condition of the integumentary system selected from one or more of acne, acne vulgaris, acne aestivalis, acne conglobata, acne cosmetic, acne fulminans, acne keloidalis nuchae, acne mechanica, acne medicamentosa, acne miliaris necrotica, acne necrotica, acne rosacea, actinic keratosis, urticaria, acute urticaria, allergic contact dermatitis, allergic dermatitis, alopecia areata, angioedema, athlete's foot, atopic dermatitis, autoeczemazation, baby acne, balding, basal cell carcinoma, bastomycosis, blackheads, birthmarks, skin pigmentation disorder, boil, bruise, bug bite, bug sting, burn, cellulitis, chiggers, chloracne, cholinergic or stress urticaria, chronic urticaria, cold type urticaria, confluent and reticulated papillomatosis, corns, cysts, dandruff, dermatitis herpetiformis, dermatographism, dyshidrotic eczema, diaper rash, dry skin, dyshidrosis, ectodermal dysplasia, hyprohidrotic ectodermal dysplasia, X-linked hyprohidrotic ectodermal dysplasia, eczema, epidermaodysplasia verruciformis, erythema nodosum, excoriated acne, exercise-induced anaphylasis folliculitis, excess skin oil, folliculitis, freckles, frostbite, fungal nails, hair density, hair growth, hair growth rate, halogen acne, hair loss, heat rash, hematoma, herpes simplex infections, hidradenitis suppurativa, hives, hyperhidrosis, hyperpigmentation, hypohidrotic ectodermal dysplasia, hypopigmentation, impetigo, ingrown hair, heat type urticaria, ingrown toenail, infantile acne or neonatal acne, itch, irritant contact dermatitis, jock itch, Kaposi's sarcoma, keloid, keratosis, keratosis pilaris, lichen planus, lichen sclerosus, lupus miliaris disseminatus faciei, melanoma, melasma, moles, molluscum contagiosum, nail growth rate, nail health, neurodermatitis, nummular eczema, occupational acne, oil acne, oily skin, onychomycosis, physical urticara, pilonidal cyst, pityriasis rosea, pityriasis versicolor, poison ivy, pomade acne, pseudofolliculitis barbae or acne keloidalis nuchae, psoriasis, psoriatic arthritis, pressure or delayed pressure urticaria, puncture wounds such as cuts and scrapes, rash, rare or water type urticaria, rhinoplasty, ringworm, rosacea, rothmund-thomson syndrome, sagging of the skin, scabis, scars, seborrhea, seborrheic dermatitis, shingles, skin cancer, skin tag, solar type urticaria, spider bite, squamous cell carcinoma, stretch marks, sunburn, tar acne, tropical acne, thinning of skin, thrush, tinea versicolor, transient acantholytic dermatosis, tycoon's cap, uneven skin tone, varicose veins, venous eczema, vibratory angioedema, vitiligo, warts, Weber-Christian disease, wound, wrinkles, x-linked hypohidrotic ectodermal dysplasia, xerotic eczema, yeast infection, and general signs and symptoms of aging.
11. The method of claim 10 , wherein the method alters, modifies, and/or changes the appearance of the integumentary system.
12. The method of claim 1 , wherein the administering is via a patch delivery system, wherein the patch delivery system comprises a composition enclosed within a membrane, and an array of delivery needles delivering about 300 ng of RNA per treatment area of about 100 cm 2 or less, or about 50 cm 2 or less, or about 20 cm 2 or less, or about 10 cm 2 or less, or about 5 cm 2 or less, or about 1 cm 2 or less, or about 0.5 cm 2 or less, or about 0.2 cm 2 or less.
13. The method of claim 1 , wherein the gene-editing protein is selected from: a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein.
14. A method for gene-editing a cell in the skin of a human subject, comprising:
(a) selecting a human patient in need of gene-editing of a skin cell;
(b) obtaining a unit dosage form of a composition comprising an RNA encoding a gene-editing protein, the unit dose form comprising about 300 ng of the RNA;
(c) administering the unit dosage form of the composition comprising the RNA encoding the gene-editing protein to the human subject, to result in the cell expressing the protein of interest, wherein the administration is intradermal, and
(d) inducing a single-strand or double-strand break in the DNA of the cell,
thereby gene-editing the cell, and wherein the unit dose of about 300 ng of the RNA encoding the gene-editing protein provides greater expression of the gene-editing protein than a dose of 1200 ng of RNA encoding the gene-editing protein during the second to fifth day after administering the RNA.
15. The method of claim 14 , wherein the RNA comprises one or more of 5-hydroxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, 5-methoxypseudouridine, and 7-deazaguanosine.
16. The method of claim 14 , wherein the RNA comprises a 5′ cap structure, a 5′-UTR comprising a Kozak consensus sequence, a 5′-UTR comprising a sequence that increases RNA stability in vivo, a 3′-UTR comprising a sequence that increases RNA stability in vivo, and/or a 3′ poly(A) tail.
17. The method of claim 14 , wherein the RNA is from about 200 nucleotides to about 5000 nucleotides in length, about 500 to about 2000 nucleotides in length, about 500 to about 1500 nucleotides in length, or about 500 to about 1000 nucleotides in length and the RNA is prepared by in vitro transcription.
18. The method of claim 14 , wherein the composition comprises one or more lipids that enhance uptake of RNA by cells.
19. The method of claim 18 , wherein the one or more lipids are selected from 3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol; 1,2-dioleoyl-3-trimethylammonium-propane; N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium; 1,2-dimyristoyl-3-trimethylammonium-propane; 1,2-dipalmitoyl-3-trimethylammonium-propane; 1,2-stearoyl-3-trimethylammonium-propane; 1,2-dioleoyl-3-dimethylammonium-propane; 1,2-dimyristoyl-3-dimethylammonium-propane; 1,2-dipalmitoyl-3-dimethylammonium-propane; 1,2-distearoyl-3-dimethylammonium-propane; dimethyldioctadecylammonium; 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine; 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine; 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine; 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine; 1,2-distearoyl-sn-glycero-3-ethylphosphocholine; 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine; 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine; 1,2-di-O-octadecenyl-3-trimethylammonium propane; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide; 2,3-dioleyloxy-N-[2-spermine carboxamide]ethyl-N,N-dimethyl-1-propanammonium trifluoroacetate; 1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamid; N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide; a transfection reagent; dioctadecyl amidoglyceryl spermine; dioleoyl phosphatidyl ethanolamine; and combinations thereof.
20. The method of claim 14 , wherein about 300 ng of RNA is administered per treatment area of about 10 cm 2 or less, or about 5 cm 2 or less, or about 1 cm 2 or less, or about 0.5 cm 2 or less, or about 0.2 cm 2 or less.
21. The method of claim 14 , wherein the composition is administered about weekly for at least 2 weeks, is administered about every other week for at least one month, is administered monthly, or is administered about every other month.
22. The method of claim 21 , wherein the composition is administered for at least two months, or at least 4 months, or at least 6 months, or at least 9 months, or at least one year.
23. The method of claim 14 , wherein the human subject has a disease, disorder, or condition of the integumentary system selected from one or more of acne, acne vulgaris, acne aestivalis, acne conglobata, acne cosmetic, acne fulminans, acne keloidalis nuchae, acne mechanica, acne medicamentosa, acne miliaris necrotica, acne necrotica, acne rosacea, actinic keratosis, urticaria, acute urticaria, allergic contact dermatitis, allergic dermatitis, alopecia areata, angioedema, athlete's foot, atopic dermatitis, autoeczemazation, baby acne, balding, basal cell carcinoma, bastomycosis, blackheads, birthmarks, skin pigmentation disorder, boil, bruise, bug bite, bug sting, burn, cellulitis, chiggers, chloracne, cholinergic or stress urticaria, chronic urticaria, cold type urticaria, confluent and reticulated papillomatosis, corns, cysts, dandruff, dermatitis herpetiformis, dermatographism, dyshidrotic eczema, diaper rash, dry skin, dyshidrosis, ectodermal dysplasia, hyprohidrotic ectodermal dysplasia, X-linked hyprohidrotic ectodermal dysplasia, eczema, epidermaodysplasia verruciformis, erythema nodosum, excoriated acne, exercise-induced anaphylasis folliculitis, excess skin oil, folliculitis, freckles, frostbite, fungal nails, hair density, hair growth, hair growth rate, halogen acne, hair loss, heat rash, hematoma, herpes simplex infections, hidradenitis suppurativa, hives, hyperhidrosis, hyperpigmentation, hypohidrotic ectodermal dysplasia, hypopigmentation, impetigo, ingrown hair, heat type urticaria, ingrown toenail, infantile acne or neonatal acne, itch, irritant contact dermatitis, jock itch, Kaposi's sarcoma, keloid, keratosis, keratosis pilaris, lichen planus, lichen sclerosus, lupus miliaris disseminatus faciei, melanoma, melasma, moles, molluscum contagiosum, nail growth rate, nail health, neurodermatitis, nummular eczema, occupational acne, oil acne, oily skin, onychomycosis, physical urticara, pilonidal cyst, pityriasis rosea, pityriasis versicolor, poison ivy, pomade acne, pseudofolliculitis barbae or acne keloidalis nuchae, psoriasis, psoriatic arthritis, pressure or delayed pressure urticaria, puncture wounds such as cuts and scrapes, rash, rare or water type urticaria, rhinoplasty, ringworm, rosacea, rothmund-thomson syndrome, sagging of the skin, scabis, scars, seborrhea, seborrheic dermatitis, shingles, skin cancer, skin tag, solar type urticaria, spider bite, squamous cell carcinoma, stretch marks, sunburn, tar acne, tropical acne, thinning of skin, thrush, tinea versicolor, transient acantholytic dermatosis, tycoon's cap, uneven skin tone, varicose veins, venous eczema, vibratory angioedema, vitiligo, warts, Weber-Christian disease, wound, wrinkles, x-linked hypohidrotic ectodermal dysplasia, xerotic eczema, yeast infection, and general signs and symptoms of aging.
24. The method of claim 23 , wherein the method alters, modifies, and/or changes the appearance of the integumentary system.
25. The method of claim 14 , wherein the administering is via a patch delivery system, wherein the patch delivery system comprises a composition enclosed within a membrane, and an array of delivery needles delivering about 300 ng of RNA per treatment area of about 100 cm 2 or less, or about 50 cm 2 or less, or about 20 cm 2 or less, or about 10 cm 2 or less, or about 5 cm 2 or less, or about 1 cm 2 or less, or about 0.5 cm 2 or less, or about 0.2 cm 2 or less.
26. The method of claim 14 , wherein the gene-editing protein is selected from: a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein.