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Last Updated: March 29, 2024

Claims for Patent: 8,211,696


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Summary for Patent: 8,211,696
Title:Method useful for HCV RNA898 infection
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.
Inventor(s): Kwong; Ann (Cambridge, MA), Byrn; Randal (Wayland, MA), Reid; Lola M. (Chapel Hill, NC)
Assignee: Vertex Pharmaceuticals Incorporated (Cambridge, MA)
Application Number:13/013,758
Patent Claims:1. A method for infecting cells with Hepatitis C Virus (HCV) comprising the step of contacting a composition comprising a cell mixture comprising liver cells and hematopoietic cells, the cells being released from the liver of a human aged three months or older after conception up to 1 year after birth, with the HCV virus RNA898 ("RNA 898" deposited on Mar. 27, 2001, in the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209; ATCC Deposit No.: PTA-3237).

2. The method according to claim 1, wherein the HCV virus is added to the composition and incubated for about 24 hours at about 37.degree. C. in a volume of about 0.52 ml per cm.sup.2 prior to washing the cells in the composition.

3. The method according to claim 1, wherein the composition further comprises a feeder cell, wherein the feeder cell is capable of providing an extracellular matrix and diffusible factors.

4. The method according to claim 1, wherein each component of the cell mixture has a size that allows it to pass through a 40-micron filter, and the cell mixture also comprises cells that express alpha fetoprotein, cells that express albumin, and cells that express glycophorin, but is free of detectable cells that express CD34 protein.

5. The method according to claim 4, wherein the presence of any CD34 protein is detected by immunofluorescence or immunoperoxidase staining.

6. The method according to claim 1, wherein each component of the cell mixture has a size that allows it to pass through a 40-micron filter, and wherein the composition further comprises an extracellular matrix or a feeder cell, wherein the feeder cell is capable of providing an extracellular matrix and diffusible factors.

7. The method according to claim 1, wherein the composition further comprises a serum-free media, said serum-free media comprising calcium, free fatty acids (FFAs), high density lipoprotein (HDL), nicotinamide, trace elements, epidermal growth factor (EGF), insulin, transferrin, hydrocortisone, and, optionally, any one of the ingredients selected from the group consisting of glucagon, liver growth factor, ethanolamine and thyrotropin releasing factor or any combination of them.

8. The method of claim 7, wherein the serum-free media does not comprise low density lipoprotein (LDL).

9. The method of claim 1, wherein the composition being prepared by the following steps: a. dissecting a liver of a human aged three months or older after conception up to 1 year after birth in a buffer comprising ethylene glycol bis (.beta.-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA); b. incubating the dissected liver in a buffer comprising collagenase to separate cells from the liver; c. passing the separated cells through a 40-micron filter; d. removing red blood cells from the filtered cells; e. resuspending the cells of step (d) in serum-free media, comprising calcium, FFAs, HDL, nicotinamide, trace elements, EGF, insulin, transferrin, hydrocortisone, and optionally, further comprising any one of the ingredients selected from the group consisting of: glucagon, liver growth factor, ethanolamine and thyrotropin releasing factor or any combination of them; and f. culturing the cells in the serum-free media of step (e).

10. The method according to any one of claims 1, 2 and 3-9, wherein the method produces more than 5,000 copies of HCV RNA seventy-two hours after administering RNA 898 to 4.times.10.sup.5 of said cells.

11. The method according to any one of claims 1, 2 and 3-9, wherein the method produces more than 10,000 copies of HCV RNA seventy-two hours after administering RNA 898 to 4.times.10.sup.5 of said cells.

12. The method according to any one of claims 1, 2 and 3-9, wherein the method produces more than 50,000 copies of HCV RNA seventy-two hours after administering RNA 898 to 4.times.10.sup.5 of said cells.

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