Compositions and methods useful for HCV infection

• US 20050287514A1
• Filed: 12/01/2004
• Published: 12/29/2005
• Est. Priority Date: 12/01/2003
• Status: Abandoned Application

• Current Assignee: Vertex Pharmaceuticals Incorporated
• Original Assignee: Vertex Pharmaceuticals Incorporated
• Inventors: Byrn, Randal
• Application Number:
  US11/001,686
• Publication Number:
  US 20050287514A1
• Time in Patent Office: Days
• US Class Current: 435/2
• CPC Class Codes: A01N 1/02 Preservation of living parts
• CPC Class Codes: A01N 1/0221 Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
• CPC Class Codes: C12N 2500/10 Metals; Metal chelators cobalamine C12N2500/38
• CPC Class Codes: C12N 2500/14 Calcium; Ca chelators; Calcitonin
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/36 Lipids
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2500/90 Serum-free medium, which may still contain naturally-sourced components
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/18 Liver cell growth factor (LCGF, Gly-His-Lys)
• CPC Class Codes: C12N 2501/335 Glucagon; Glucagon-like peptide [GLP]; Exendin
• CPC Class Codes: C12N 2501/70 Enzymes
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 5/067 Hepatocytes

Abstract

The present invention provides compositions comprising cells that can effectively produce HCV after HCV infection, compositions for culturing the cells, methods for making the composition and methods for infecting the cells in the composition with HCV. The present invention also provides methods for assaying HCV production and methods for evaluating compounds that affect the production of HCV.

Claims

1. A composition comprising a cryopreserved cell mixture that comprises liver cells and hematopoietic cells isolated from the liver of a human aged three months or older after conception, wherein a preparation that comprises 4×

10⁴ thawed cells of said cryopreserved cell mixture in the presence of a feeder cell line in a growth media produces more than about 5000 copies of hepatitis C virus (HCV) RNA in the media seventy two hours after administration of HCV virus RNA898 to said preparation.

27. The composition according claim 1, further comprising an HCV.

28. The composition according to claim 27, wherein the HCV is RNA898.

39. A method for infecting cells with HCV comprising the step of contacting the composition of any one of claims 1 to 29 or the cell mixture prepared according to claim 31 with the HCV virus RNA898 or its infectious equivalent.

40. The method according to claim 39, wherein the HCV virus is added to composition and incubated for about 24 hours at about 37°

C. in a volume of about 0.52 ml per cm² prior to washing the cells in the composition.

41. A method for assaying HCV infection comprising the steps of:

(a) incubating the composition according to claim 1, with a feeder cell;

(b) contacting the cells in the composition with RNA898 or its infectious equivalent; and

(c) measuring the presence of the HCV RNA associated with the cells of the composition, the media in which the cells are cultured or both the cells and the media.

42. The method according to claim 41, wherein the feeder cell is the STO(Reid 99) cell.

43. The method according to claim 41, wherein the quantity of HCV RNA is measured by comparing (a) the amount of HCV RNA present associated with the cells or media in which the cells are cultivated with (b) an amount of RNA from a second virus that is used as an internal control.

44. The method according to claim 43, wherein the second viral RNA is from Bovine Viral Diarrhea Virus (BVDV).

45. A method for evaluating the ability of a compound to affect the production of an HCV comprising the steps of:

(a) incubating the composition according to claim 1, with a feeder cell;

(b) contacting the cells in the composition with RNA898;

(c) administering the compound to the composition before or after the cells are contacted with RNA898 or its infectious equivalent; and

(d) measuring the HCV associated with the cells, the media in which the cells are cultured or both the cells and the media.

46. The method according to claim 45, wherein the compound inhibits HCV production.

47. The method according to claim 45, wherein a plurality of compounds are screened simultaneously for their ability to inhibit HCV production.

48. The method according to claim 45, comprising the further step of comparing the measurement of step (d) with the amount of HCV associated with control cells, the media in which the control cells are cultured or both the control cells and the media, wherein the control cells have been subjected to steps (a)-(d) except that no compound or a known inactive compound has been administered.

49. The method according to claim 45, wherein the feeder cell is the STO(Reid 99) cell.

50. The method according to claim 45, wherein the presence of HCV is measured by determining the quantity of HCV RNA associated with the cells or media in which the cells are cultivated.

51. The method according to claim 45, wherein the quantity of HCV RNA is determined by reverse transcriptase polymerase chain reaction (RT PCR).

52. The method according to claim 45, wherein the quantity of HCV RNA is measured by comparing (a) the amount of HCV RNA associated with the cell or the media in which the cells are cultivated with (b) an amount of RNA from a second virus that is used as an internal control.

53. The method according to claim 45, wherein the second virus is Bovine Viral Diarrhea Virus (BVDV).

2. (canceled)

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5. (canceled)

6. A composition comprising a cell mixture prepared by the following steps:

(a) obtaining a suspension of liver cells from a human aged three months or older after conception in a buffer comprising EGTA by treating said liver cells with a protease, wherein said suspension of liver cells has been treated to remove objects that are 40 micron or greater and said suspension of liver cells does not contain red blood cells;

(b) resuspending the cells of step (a) in a media comprising BSA, nicotinamide, epidermal growth factor (EGF), insulin, transferrin and hydrocortisone and optionally, any one of the ingredients selected from the group consisting of glucagon, liver growth factor, ethanolamine and thyrotropin releasing factor; and

(c) culturing the cells in the serum free media of step (b).

7. The method of claim 6 further comprising:

(d) cryopreserving the cells of step (a) in suspension that comprises 10% DMSO and 10% fetal calf serum prior to step (b); and

(e) thawing the composition of step (d) prior to the resuspension in step (b).

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12. A composition comprising a cell mixture prepared by the following steps:

(a) dissecting a liver of a human aged three months or older after conception in a buffer comprising EGTA;

(b) incubating the dissected liver in a buffer comprising collagenase to separate cells from the liver;

(c) remove objects 40 micron or greater from the separated cells;

removing red blood cells from the separated cells;

(e) resuspending the cells in 10% DMSO and 10% fetal calf serum;

(f) cryopreserving the composition of step 5;

(g) thawing the composition of step 6;

(h) resuspending the cells of step (f) in a serum free media, comprising calcium, free fatty acids (FFAs), high density lipoprotein (HDL), nicotinamide, trace elements, epidermal growth factor (EGF), insulin, transferrin and hydrocortisone and optionally, any one of the ingredients selected from the group consisting of glucagon, liver growth factor, ethanolamine and thyrotropin releasing factor; and

(i) culturing the cells in the serum free media of step (e).

13. A composition comprising a cell mixture prepared by the following steps:

(a) dissecting a liver of a human aged three months or older after conception in a buffer comprising EGTA;

(b) incubating the dissected liver in a buffer comprising collagenase to separate cells from the liver;

(c) remove objects 40 micron or greater from the separated cells;

(d) removing red blood cells from the separated cells;

(e) resuspending the cells of step (d) in a media comprising BSA, nicotinamide, epidermal growth factor (EGF), insulin, transferrin and hydrocortisone and optionally, any one of the ingredients selected from the group consisting of glucagon, liver growth factor, ethanolamine and thyrotropin releasing factor; and

(f) culturing the cells in the serum free media of step (e).

14. A composition according to claim 13, comprising the further steps between steps (d) and (e):

(g) resuspending the cells of step (d) in 10% DMSO and 10% fetal calf serum;

(h) cryopreserving the composition of step g; and

(i) thawing the composition of step h.

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17. (canceled)

18. A composition comprising an expanded mixed cell population of liver cells and hematopoietic cells released from the liver of a human aged three months or older after conception, wherein said mixed cell population is selected to exclude cells that express CD34+ but includes cells that express alpha fetoprotein, albumin, and glycophorin.

19. (canceled)

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26. (canceled)

29. A composition comprising a media, BSA, nicotinamide, EGF, insulin, transferrin, and hydrocortisone, and, optionally, not low density lipoprotein (LDL) and, optionally, not any one of the ingredients selected from the group consisting of glucagon, liver growth factor, ethanolamine and thyrotropin releasing factor.

30. (canceled)

31. A method for isolating and cultivating a cell mixture comprising the steps of:

(a) obtaining a suspension of liver cells from a human aged three months or older after conception in a buffer comprising EGTA by treating said liver cells with a protease, wherein said suspension of liver cells has been treated to remove objects that are 40 micron or greater and said suspension of liver cells does not contain red blood cells;

(b) resuspending the cells of step (a) in a media comprising BSA, nicotinamide, EGF, insulin, transferrin, hydrocortisone; and

(c) culturing the cells in the serum free media of step (b).

32. The method according to claim 31, comprising the further steps between steps a) and b):

(d) cryopreserving the cells of the suspension of step (a) in a composition comprising 10% DMSO and 10% fetal calf serum; and

(e) thawing the cryopreserved cells of step (d) prior to step (b).

33. The method of claim 31, wherein said suspension of liver cells are obtained in step (a) by dissecting a liver of a human aged three months or older after conception in a buffer comprising EGTA.

34. The method of claim 33, wherein said buffer comprising EGTA is supplemented or replaced with a protease containing buffer wherein said protease is selected from the group consisting of collagenase, trypsin, pronase, and dipase.

35. The method of claim 33, said buffer comprising EGTA is supplemented or replaced with a protease containing buffer wherein said buffer comprises a cocktail of two or more proteases selected from the group consisting of collagenase, trypsin, pronase, and dipase.

36. The method of claim 34 wherein said buffer comprising EGTA further comprises an enzyme to breakdown the carbohydrate components of the tissue, said enzyme selected from the group consisting of hyaluronidase, and neuraminadase.

37. The method according to claim 31, wherein the red blood cells are removed by centrifuging the suspended cells at low speed (50×

g) to pellet the larger cells for approximately 3 to 4 minutes, washing the cells that pellet and repeating the centrifuging and washing steps.

38. The method according to claim 31, wherein the media does not contain low density lipoprotein (LDL);

and/or media does not contain calcium, FFA, HDL, or trace elements; and

/or media does not contain glucagon, liver growth factor, ethanolamine or thyrotropin releasing factor; and

/or the media is not serum free media.

54. A method of preparing a mixed population of liver cells comprising:

(a) obtaining a suspension of liver cells from a human aged three months or older after conception in a buffer comprising EGTA by treating said liver cells with a protease, wherein said suspension of liver cells has been treated to remove objects that are 40 micron or greater and said suspension of liver cells does not contain red blood cells;

(b) isolating CD34−

/alpha fetoprotein+/glycoprotein+/albumin+cells from said suspension of liver cells;

(c) resuspending the cells of step (b) in a media comprising BSA, nicotinamide, epidermal growth factor (EGF), insulin, transferrin and hydrocortisone and optionally, any one of the ingredients selected from the group consisting of glucagon, liver growth factor, ethanolamine and thyrotropin releasing factor; and

(d) co-culturing said cells from step (c) in culture media comprising feeder cells to expand said population of liver cells.

55. The method of claim 54 comprising cryopreserving the cells of the suspension of step (a) or the cells isolated after step (b) in a composition comprising 10% DMSO and 10% fetal calf serum;

and thawing said cryopreserved cells prior to step (c).

56. (canceled)