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1. WO2020112979 - ÉDITION DE GÈNE THÉRAPEUTIQUE POUR UNE MALADIE ASSOCIÉE À ELANE

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

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CLAIMS

1. A modified synthetic nucleic acid comprising a nucleic acid sequence shown in Table 1, SEQ ID NOS: 1-374, wherein there is at least one chemical modification to a nucleotide in the nucleic acid molecule.

2. The modified synthetic nucleic acid of claim 1, wherein the at least one chemical modifications is located at one or more terminal nucleotides in nucleic acid molecule.

3. The modified synthetic nucleic acid of claim 2, wherein the at least one chemical modification is selected from the group consisting of 2'-0-methyl 3'phosphorothioate (MS), 2'-0-methyl-3'- phosphonoacetate (MP), 2'-0-Ci-4alkyl, 2'-H, 2'-0-Ci.3alky]-0-Ci.3alkyl, 2'-F, 2'-NH2, 2'- arabino, 2'- F-arabino, 4'-thioribosyl, 2-thioU, 2-thioC, 4-thioU, 6-thioG, 2-aminoA, 2- aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7- deazaadenine, 7-deaza-8-azaadenine, 5-methylC, 5-methylU, 5-hydroxymethylcytosine, 5- hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine, 5- propynyluracil, 5- ethynylcytosine, 5-ethynyluracil, 5-allylU, 5-allylC, 5-aminoallyl-uracil, 5-aminoallyl- cytosine, an abasic nucleotide ("abN"), Z, P, UNA, isoC, isoG, 5 -methyl -pyrimidine, x(A,G,C,T) and y(A,G,C,T), a phosphorothioate intemucleotide linkage, a phosphonoacetate intemucleotide linkage, a thiophosphonoacetate intemucleotide linkage, a methylphosphonate intemucleotide linkage, a boranophosphonate intemucleotide linkage, a phosphorodithioate intemucleotide linkage, 4'-thioribosyl nucleotide, a locked nucleic acid ("LNA") nucleotide, an unlocked nucleic acid ("ULNA") nucleotide, an alkyl spacer, a heteroalkyl (N, O, S) spacer, a 5'- and/or 3'-alkyl terminated nucleotide, a Unicap, a 5'- terminal cap known from nature, an xRNA base

(analogous to "xDNA" base), an yRNA base (analogous to "yDNA" base), a PEG substituent, and a conjugated linker to a dye or non-fluorescent label (or tag).

4. The modified synthetic nucleic acid of any one of claims 1-3, wherein the at least one chemical modification is located only at the 3’ end, or added only at the 5’ end, or added at both the 5' and 3' ends of the synthetic nucleic acid molecule.

5. The modified synthetic nucleic acid of any one of claims 1-4, wherein the at least one chemical modification is located to first three nucleotides and to the last three nucleotides of the synthetic nucleic acid molecule.

6. The modified synthetic nucleic acid of any one of claims 1-5, wherein the modified synthetic nucleic acid sequence further comprising a crRNA/tracrRNA sequence.

7. The modified synthetic nucleic acid of any one of claims 1-6, wherein the modified synthetic nucleic acid is a single guide RNA (sgRNA).

8. The modified synthetic nucleic acid of any one of claims 1-7 for use in the ex vivo targeted genome editing of the ELANE gene in a progenitor cell purpose.

9. The modified synthetic nucleic acid of any one of claims 1-7 for use in an ex vivo method of producing a progenitor cell or a population of progenitor cells wherein the cells or the differentiated progeny therefrom have decreased ELANE mRNA or protein expression.

10. The modified synthetic nucleic acid of any one of claims 1-7 for use in an ex vivo method of producing an isolated genetic engineered human cell or a population of genetic engineered human cells having at least one genetic modification.

11. The modified synthetic nucleic acid of any one of claims 8-10, wherein the modified synthetic nucleic acid is used in combination with a DNA-targeting endonuclease Cas (CRISPR- associated) protein in a ribonucleoprotein (RNP) complex.

12. The modified synthetic nucleic acid of claim 11, wherein the Cas protein is Cas 9.

13. The modified synthetic nucleic acid of claim 11 or 12, wherein the RNP complex is used in the electroporation of cells.

14. The modified synthetic nucleic acid of claim 13, wherein the step of electroporation is performed in a solution comprising glycerol.

15. The modified synthetic nucleic acid of claim 10, wherein the isolated human cell is a

hematopoietic progenitor cell or a hematopoietic stem cell.

16. The modified synthetic nucleic acid of claim 15, wherein the hematopoietic progenitor is a cell of the erythroid lineage.

17. The modified synthetic nucleic acid of claim 10, wherein the isolated human cell is an induced pluripotent stem cell.

18. The modified synthetic nucleic acid of any one of claims 8-17, wherein the progenitor cell or ascquires at least one genetic modification.

19. The modified synthetic nucleic acid of claim 18, wherein the at least one genetic modification is a deletion, insertion or substitution of the genetic sequence of the cell.

20. A composition comprising a modified synthetic nucleic acid molecule of any one of claims 1-7

21. The composition of claim 20, further comprising a DNA-targeting endonuclease Cas (CRISPR- associated) protein.

22. The composition of claim 21, wherein the Cas protein is Cas 9.

23. The composition of any one of claims 21-22 for use in an ex vivo method of producing a

progenitor cell or a population of progenitor cells wherein the cells or the differentiated progeny therefrom have decreased ELANE mRNA or protein expression.

24. The composition of any one of claims 21-22 for use in an ex vivo method of producing an

isolated genetic engineered human cell or a population of progenitor cells having at least one genetic modification.

25. The composition of any one of claims 21-22, wherein the composition is used in the

electroporation of cells.

26. The composition of claim 31, wherein the step of electroporation is performed in a solution comprising glycerol.

27. A ribonucleoprotein (RNP) complex comprising a DNA-targeting endonuclease Cas (CRISPR- associated) protein and a modified synthetic nucleic acid molecule of any one of claims 1-7.

28. The RNP complex of claim 27 for use in an ex vivo method of producing a progenitor cell or a population of progenitor cell wherein the cells or the differentiated progeny thereof have decreased ELANE mRNA or protein expression.

29. The RNP complex of claim 27 for use in an ex vivo method of producing an isolated genetic engineered human cell or a population of genetic engineered human cells having at least one genetic modification.

30. The RNP complex of any one of claims 27-29, wherein the RNP complex is used in the

electroporation of cells.

31. The RNP complex of claim 30, wherein wherein the step of electroporation is performed in a solution comprising glycerol.

32. A method for producing a progenitor cell or a population of progenitor cells having decreased ELANE mRNA or protein expression, the method comprising contacting an isolated progenitor cell with an effective amount of a modified synthetic nucleic acid of any of claims 1-7, a composition of any one of claims 20-26, or a ribonucleoprotein (RNP) complex of claim 27, whereby the contacted cells or the differentiated progeny cells therefrom have decreased ELANE mRNA or protein expression.

33. The method of claim 32, wherein the at least one genetic modification is a deletion, insertion or substitution of the genetic sequence of the cell.

34. The method of any one of claims 32, wherein the isolated progenitor cell is a hematopoietic progenitor cell or a hematopoietic stem cell.

35. The method of claim 34, wherein the hematopoietic progenitor is a cell of the erythroid lineage.

36. The method of any one of claims 32, wherein the isolated progenitor cell is an induced

pluripotent stem cell.

37. The method of any one of claims 32-36, wherein the isolated progenitor cell is contacted ex vivo or in vitro.

38. The method of claim 37, wherein the contacted progenitor cell acquires at least one genetic

modification.

39. The method of any one of claims 37-38, wherein the contacted progenitor cell or contacted cell are further electroporated.

40. The method of claim 39, wherein the step of electroporation is performed in a solution

comprising glycerol.

41. A population of genetically edited human cells having at least one genetic modification resulting in decreased ELANE mRNA or protein expression.

42. The population of claim 41, wherein the genetically edited human cells are isolated.

43. A population of genetically edited progenitor cells having at least one genetic modification

resulting in decreased ELANE mRNA or protein expression.

44. A composition comprising isolated genetically edited human cells of claims 41 and 42.

45. A composition comprising genetically edited progenitor cells of claims 43.

46. A method of treating a disease associated with abnormal ELANE gene expression, the method comprising, administering to a subject in need thereof a gene editing agent that targets the ELANE gene, wherein the edited ELANE gene comprises at least one mutation selected from the group consisting of a loss-of-function mutation, a through stop-gain mutation, a frameshift mutation, a premature termination codon, and a splicing mutation that encodes a premature termination codon.

47. The method of claim 47, wherein the disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD) (including acquired disease or genetic alpha- 1 antitrypsin deficiency), cystic fibrosis, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), asthmatic conditions, rheumatoid arthritis, chronic kidney disease, cyclic neutropenia, and severe congential neutropenia.

48. The method of claims 46 or 47, wherein the gene editing agent is selected from the group

consisting of clustered regularly interspaced short palindromic repeats (CRISPR), Transcription activator-like effector nucleases (TALEN), Meganuclease, Zinc finger nucleases, Homologous recombination, and deaminase fusion proteins.

49. The method of claim 46, wherein the at least one mutation results in induction of nonsense- mediated decay, translational repression, or knockdown of transcript.

50. The method of claim 46, wherein the abnormal ELANE gene expression results in abnormal neutrophil elastase protein.

51. The method of claim 46, wherein the gene editing agent is a modified synthetic nucleic acid of any of claims 1-7, a composition of any one of claims 20-26, or a ribonucleoprotein (RNP) complex of claim 27.