Pluripotent cell lines and methods of use thereof

• US 20100167286A1
• Filed: 06/24/2009
• Published: 07/01/2010
• Est. Priority Date: 06/24/2008
• Status: Active Grant

• Current Assignee: Parkinson's Institute, Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Parkinson's Institute, Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Byrne, James Anthony, Schüle, Birgitt, Nguyen, Ha Nam, Cord, Brandon John, Reijo Pera, Renee Ann, Palmer, Theodore D., Byers, Blake, Langston, J. William
• Granted Patent: US 8,669,048 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2501/50 Cell markers; Cell surface determinants
• CPC Class Codes: C12N 2501/602 Sox-2
• CPC Class Codes: C12N 2501/603 Oct-3/4
• CPC Class Codes: C12N 2501/604 Klf-4
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2501/71 Oxidoreductases (EC 1.)
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/1307 from adult fibroblasts
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2740/15032 Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• CPC Class Codes: C12N 5/0619 : Neurons
• CPC Class Codes: C12N 7/00 : Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/136 : Screening for pharmacological compounds
• CPC Class Codes: G01N 2333/47 : Assays involving proteins of known structure or function as defined in the subgroups
• CPC Class Codes: G01N 33/5023 : on expression patterns
• CPC Class Codes: G01N 33/5058 : Neurological cells

Abstract

Methods of generating cell lines with a sequence variation or copy number variation of a gene of interest, methods of use thereof, and cell lines with a sequence variation or copy number variation of a gene of interest are provided.

Claims

1. A method of producing dopaminergic cells from a patient having a diagnosis of Parkinson'"'"'s disease or Parkinson'"'"'s-like disease, the method comprising:

a. obtaining fibroblasts from the patient;

b. dedifferentiating the fibroblasts; and

c. differentiating the stem cells towards a dopaminergic cell fate.

3. The method of claim 1 wherein the stem cells are differentiated towards a midbrain dopaminergic cell fate.

4. The method of claim 1 wherein dedifferentiating the fibroblasts induces pluripotency.

5. The method of claim 1 wherein the patient carries a genetic variation or mutation known to be associated with Parkinson'"'"'s disease or Parkinson'"'"'s-like disease.

6. The method of claim 5 wherein the genetic variation of interest is a copy number variation of a gene of interest.

7. The method of claim 6, wherein the cell line has three copies of the gene of interest.

10. The method of claim 7 wherein the gene of interest is PARK1 (SNCA or α

-synuclein).

11. The method of claim 6 wherein the gene of interest is PARKS (LRRK2).

12. The method of claim 11 wherein the patient carries a homozygous mutation of LRRK2.

13. The method of claim 12 wherein the mutation comprises a G2019S mutation.

14. The method of claim 1 wherein the patient has Parkinson'"'"'s disease.

2. (canceled)

8. (canceled)

9. (canceled)

15. (canceled)

16. A method of determining whether an agent is useful in the treatment of Parkinson'"'"'s disease or Parkinson'"'"'s-related disease, the method comprising:

a. contacting dopaminergic cells produced from induced pluripotent stem cells derived from a patient with Parkinson'"'"'s disease or Parkinson'"'"'s-like disease;

b. detecting a response in the cells; and

c. determining whether the agent is useful in the treatment based on the detected response.

17. The method of claim 16 wherein the detected response is a change in cell viability, cellular chemistry, cellular function, mitochondrial function, cell aggregation, cell morphology, cellular protein aggregation, gene expression, cellular secretion, or cellular uptake.

18. The method of claim 16 wherein the agent is selected from a small molecule, a drug, an antibody, a hybrid antibody, an antibody fragment, a siRNA, an antisense RNA, an aptamer, a protein, or a peptide.

19. The method of claim 16 wherein the agent corrects an α

-synuclein dysfunction.

20. The method of claim 19 wherein the agent is selected from the group consisting of apomorphine, pyrogallol, 1,4-naphthoquinone, cisplatin, isoproterenol, pyrogallin, cianidanol, sulfasalazine, quinalizarin, benserazide, hexachlorophene, pyrvinium pamoate, dobutamine, methyl-dopa, curcumin, berberine chloride, daidzein, merbromin, norepinephrine, dopamine hydrochloride, carbidopa, ethylnorepinephrine hydrochloride, tannic acid, elaidyphosphocholine, hydroquinone, chlorophyllide Cu complex Na salt, methyldopa, isoproterenol hydrochloride, benserazide hydrochloride, dopamine, dobutamine hydrochloride, thyroid hormone, purpurin, sodium beta-nicotinamide adenine dinucleotide phosphate, lansoprazole, dyclonine hydrochloride, pramoxine hydrochloride, azobenzene, cefamandole sodium, cephaloridine, myricetin, 6,2′

,3′

-trihydroxyflavone, 5,7,3′

,4′

,5′

-pentahydroxyflavone, 7,3′

,4′

,5′

-tetrahydroxyflavone, (5,6,7,4′

-tetrahydroxyflavone), baicalein, eriodictyol, 7,3′

,4′

-trihydroxyisoflavone, epigallocatechin gallate, quercetin, gossypetin (3,5,7,8,3′

,4′

-hexahydroxyflavone), 2′

,3′

-dihydroxyflavone, 3′

,4′

-dihydroxyflavone, 5,6-dihydroxy-7-methoxyflavone, baicalein-7-methyl ether, 1-dopa, DOPAC, homogentisic acid, 6-hydroxydopamine, epinephrine, 3,4-dihydroxycinnamic acid, 2,3-dihydroxynaphthalene, 3,4-dihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid, 1,2,3-trihydroxybenzoic acid, gallate (gallic acid), benzoquinone, catechol, rifampicin, rosmarinic acid, baicalin, tanshinones I and II, emodin, procyanidin B4, resveratrol, rutin, fisetin, luteolin, fustin, epicatechin gallate, catechin, alizarin, tannic acid, eriodyctol, carboplatin, purpurogallin-4-carboxylic acid, koparin, 2,3,4-trihydroxy-4′

-ethexybenzophenone, baeomycesic acid, hamtoxylin, iriginol hexaaceatate, 4-acetoxyphenol, theaflavin monogallate, theaflavin digallate, stictic acid, purpurogallin, 2,5-dihydroxy-3,4-dimethoxy-4′

-ethoxybenzophenone, promethazine hydrochloride, oxidopamine hydrochloride, pyrantel pamoate, elaidylphosphocholine, amphotericin B, gallic acid, fumarprotocetraric acid, theaflavin, haematoxylin pentaacetate, 4-methoxydalbergione, epigallocatechin-3-monogallate, rolitetracycline, 7,3′

-dimethoxyflavone, liquiritigenin dimethyl ether, catechin pentaacetate, apigenin, 3,4-dedesmethyl-5-deshydroxy-3′

-ethoxyscleroin, derivatives and analogs thereof.

21. The method of claim 16 wherein the induced pluripotent stem cells are derived from human fibroblasts.

23. The method of claim 16 wherein the induced pluripotent stem cells are produced with a method comprising the use of three factors.

24. The method of claim 23 wherein the three factors are OCT4, SOX2, and KLF4.

25. The cell line of claim 16, wherein the induced pluripotent stem cells are further differentiated to adopt a midbrain dopaminergic cell fate.

26. The cell line of claim 25 wherein the induced pluripotent stem cells are differentiated to adopt the cell fate in about 20 days.

27. The method of claim 16 wherein the patient carries a genetic variation or mutation known to be associated with Parkinson'"'"'s disease or Parkinson'"'"'s-like disease.

28. The method of claim 27 wherein the genetic variation of interest is a copy number variation of a gene of interest.

29. The method of claim 28, wherein the cell line has three copies of the gene of interest.

32. The method of claim 29 wherein the gene of interest is PARK1 (SNCA or α

-synuclein).

36. The method of claim 16 wherein the patient has Parkinson'"'"'s disease.

22. (canceled)

30. (canceled)

31. (canceled)

33. The method of claim 31 wherein the gene of interest is PARK8 (LRRK2).

34. The method of claim 33 wherein the patient carries a homozygous mutation of LRRK2.

35. The method of claim 34 wherein the mutation comprises a G2019S mutation.

37. (canceled)

38. An induced pluripotent cell line from a patient with Parkinson'"'"'s disease or Parkinson'"'"'s-like disease, produced by:

a. obtaining fibroblasts from a patient with Parkinson'"'"'s disease or Parkinson'"'"'s-like disease carrying a genetic mutation which causes the disease;

b. dedifferentiating the fibroblasts, whereby producing induced pluripotent stem cells; and

c. differentiating the stem cells towards a dopaminergic cell fate, whereby producing an induced pluripotent cell line from a patient with Parkinson'"'"'s disease or Parkinson'"'"'s-like disease.

40. The cell line of claim 38 wherein the pluripotent stem cells are differentiated towards a midbrain dopaminergic cell fate.

41. The cell line of claim 38 wherein the patient carries a genetic variation or mutation known to be associated with Parkinson'"'"'s disease or Parkinson'"'"'s-like disease.

42. The cell line of claim 41 wherein the genetic variation of interest is a copy number variation of a gene of interest.

43. The cell line of claim 42, wherein the cell line has three copies of the gene of interest.

46. The cell line of claim 43 wherein the gene of interest is PARK1 (SNCA or α

-synuclein).

47. The cell line of claim 42 wherein the gene of interest is PARK8 (LRRK2).

50. The cell line of claim 38 wherein the patient has Parkinson'"'"'s disease.

52. The cell line of claim 38 wherein the induced pluripotent cell line is produced without the use of a retrovirus or a lentivirus.

53. The cell line of claim 38 wherein the induced pluripotent cell line is produced with a method comprising the use of three factors.

54. The cell line of claim 53 wherein the three factors are OCT4, SOX2, and KLF4.

55. The cell line of claim 38 wherein the induced pluripotent cell line is differentiated to adopt the cell fate in about 20 days.

59. The cell line of claim 50 wherein the induced pluripotent cell line is produced from fibroblasts of a patient with Parkinson'"'"'s disease.

39. (canceled)

44. (canceled)

45. (canceled)

48. The cell line of 47 wherein the patient carries a homozygous mutation of LRRK2.

49. The cell line of claim 48 wherein the mutation comprises a G2019S mutation.

51. (canceled)

56. A human induced pluripotent cell line from a patient with Parkinson'"'"'s disease wherein the cell line comprises a genetic variation related to Parkinson'"'"'s disease.

57. The cell line of claim 56 wherein the variation is triplication of the SNCA gene.

58. The cell line of claim 56 wherein the variation is a homozygous mutation of the LRRK2 gene.

63. The cell line of claim 56, wherein the induced pluripotent cell line is further differentiated to adopt a midbrain dopaminergic cell fate.

64. The cell line of claim 63 wherein the induced pluripotent cell line is differentiated to adopt the cell fate in about 20 days.

60. (canceled)

61. (canceled)

62. (canceled)

65. A human induced pluripotent cell line wherein the cell line comprises a genetic variation in the SNCA gene or the LRRK2 gene.