ENGINEERED NUCLEIC ACIDS AND METHODS OF USE THEREOF FOR NON-HUMAN VERTEBRATES
Provided are formulations, compositions, kits and methods for delivering biological moieties such as modified nucleic acids into cells to induce, reduce or modulate protein expression in non-human vertebrates.
1 . A method of producing a polypeptide of interest in a cell, tissue or bodily fluid of a non-human vertebrate subject in need thereof comprising administering to said subject a pharmaceutical composition comprising a nucleic acid encoding said polypeptide of interest.
2 . The method of claim 1 , wherein the pharmaceutical composition is formulated.
3 . The method of claim 2 , wherein the formulation is selected from saline or a lipid formulation.
4 . The method of claim 2 , wherein the formulation is administered by a route selected from the group consisting of intravenous, intramuscular, subcutaneous, and local.
5 . The method of claim 1 , wherein the pharmaceutical composition is administered on a schedule selected from the group consisting of three times a day, twice a day, once a day, every other day, every third day, weekly, biweekly, every three weeks, every four weekly, and monthly.
6 . The method of claim 5 , wherein the formulation is administered by multiple administrations.
7 . The method of claim 1 , wherein the non-human vertebrate is selected from the group consisting of alpaca, banteng, bison, camel, cat, cattle, deer, dog, donkey, elk, gayal, goat, guinea pig, horse, llama, mouse, mule, pig, rabbit, rat, reindeer, sheep water buffalo, yak, caiques, canary, cattle egret, chicken, cockatiel, cockatoo, conure, dove, duck, finch, geese, lovebird, macaw, parakeet, parrot, parrotlet, pigeon, pionus, rosella, turkey, iguana, lizard, snake, turtle, tortoise, caecilian, frog, newt, salamander, and toad.
8 . The method of claim 7 , wherein the non-human vertebrate is a mouse.
9 . The method of claim 8 , wherein the mouse is selected from the group consisting of a transgenic mouse, a knock-in mouse and a knock-out mouse.
10 . The method of claim 1 , wherein the bodily fluid is selected from the group consisting of peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (C SF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood.
11 . The method of claim 1 , wherein the tissue is selected from the group consisting of liver, spleen, kidney, lung, heart, peri-renal adipose tissue, thymus and muscle.
12 . The method of claim 1 , wherein the polypeptide of interest is selected from the group consisting of insulin, feline interferon, erythropoietin, cyclosporine, Thymosin Beta-4, arginine vasopressin, bovine somatotropin, oxytocin, ghrelin, gonadorelin, pregnant mare serum gonadotrophin (PMSG), equine chorionic gonadotrophin (ECG), human chorionic gonadotrophin (hCG), gonadotrophin-releasing hormone analog (GRHa), pancreatic enzymes, Cre recombinase, an insulin-like growth factor, hGH, tPA, Interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega, IFN tau, tumor necrosis factor (TNF) alpha, TNF beta, TNF gamma, TRAIL, G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.
13 . The method of claim 12 , wherein the polypeptide of interest is selected from the group consisting of insulin, feline interferon, erythropoietin, cyclosporine, Thymosin Beta-4, arginine vasopressin, bovine somatotropin, oxytocin, ghrelin, gonadorelin, pregnant mare serum gonadotrophin (PMSG), equine chorionic gonadotrophin (ECG), human chorionic gonadotrophin (hCG), gonadotrophin-releasing hormone analog (GRHa), pancreatic enzymes and Cre recombinase.
14 . The method of claim 1 , wherein the nucleic acid comprises one or more modifications selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-i sopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.
15 . A kit for producing a first polypeptide of interest in a cell, tissue or bodily fluid of a non-human vertebrate subject in need thereof comprising a first nucleic acid encoding said first polypeptide of interest.
16 . The kit of claim 15 , further comprising a second nucleic acid encoding a second polypeptide of interest.
17 . The kit of claim 16 where the second polypeptide of interest is different than the first polypeptide of interest.
18 . The kit of claim 15 , wherein the non-human vertebrate is selected from the group consisting of alpaca, banteng, bison, camel, cat, cattle, deer, dog, donkey, elk, gayal, goat, guinea pig, horse, llama, mouse, mule, pig, rabbit, rat, reindeer, sheep water buffalo, yak, caiques, canary, cattle egret, chicken, cockatiel, cockatoo, conure, dove, duck, finch, geese, lovebird, macaw, parakeet, parrot, parrotlet, pigeon, pionus, rosella, turkey, iguana, lizard, snake, turtle, tortoise, caecilian, frog, newt, salamander, and toad.
19 . The kit of claim 15 , wherein the polypeptide of interest is selected from the group consisting of insulin, feline interferon, erythropoietin, cyclosporine, Thymosin Beta-4, arginine vasopressin, bovine somatotropin, oxytocin, ghrelin, gonadorelin, pregnant mare serum gonadotrophin (PMSG), equine chorionic gonadotrophin (ECG), human chorionic gonadotrophin (hCG), gonadotrophin-releasing hormone analog (GRHa), pancreatic enzymes, Cre recombinase, an insulin-like growth factor, hGH, tPA, Interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega, IFN tau, tumor necrosis factor (TNF) alpha, TNF beta, TNF gamma, TRAIL, G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.
20 . The kit of claim 15 , wherein the nucleic acid comprises one or more modifications selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-i sopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.