Multicellular living organisms and unmodified parts thereof and – Nonhuman animal
Reexamination Certificate
2000-07-12
2003-12-09
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
C424S093100, C530S388100, C530S388150, C530S388200
Reexamination Certificate
active
06660905
ABSTRACT:
TECHNICAL FIELD
The field of this invention is mammals comprising xenogeneic tissue, and in particular xenogeneic hepatocellular tissue.
BACKGROUND
The liver is a critically important organ for monitoring and adjusting plasma constituents. Hepatocytes are active in controlling levels of blood glucose, lipids and cholesterol, and a number of plasma proteins, including albumin, fibrinogen and prothrombin, and several complement factors. The structure of a liver lobule is that of a hexagon with portal triads at each corner, where each triad contains branches of the hepatic portal vein, hepatic artery and bile duct, so that each hepatocyte is in a close association with the vascular system.
Hepatocytes synthesize triglycerides, cholesterol and phospholipids. Much of the lipid synthesized is then packaged with proteins and released into the circulation as VLDLs, providing a source of fatty acids for all cells. Hepatocytes also synthesize the enzyme essential for formation of cholesterol esters in HDL, remove chylomicron fragments from the circulation, and are an indirect source of LDLs, which are formed in plasma from VLDLs depleted of fatty acids. Balancing the lipoprotein levels and cholesterol content in the circulation has proven to be a critical factor in vascular disease.
Glucose from the blood is stored by hepatocytes in the form of glycogen, which is a major source of glucose for other cells in the body. During meals with high glucose, insulin increases the ability of hepatocytes to synthesize glycogen. As blood glucose drops, glucagon and epinephrine increase the ability of hepatocytes to degrade glycogen. Enzyme deficiencies associated with glycogen deficiencies can result in storage diseases. The liver also has other specialized function other than glucose storage, including: detoxification; synthesis of critical plasma proteins, such as coagulation proteins, alpha-1 antitrypsin, and albumin; amino acid and ammonia metabolism; heme synthesis; and vitamin and cofactor biosynthesis.
Despite its specialized functions, the liver has a unique regenerative capacity. After partial hepatectomy, the liver mass is restored by division of fully differentiated hepatocytes. Even in adults, these cells have a tremendous replicative ability. The existence of liver stem cells remains controversial, but such cells may be active in liver growth after severe injury.
The response of hepatocytes to tissue damage is mediated by several cytokines. Immediately after an injury, hepatocytes undergo a priming phase in which they become competent to enter the cell cycle. This phase is characterized by expression of the proto-oncogenes c-myc and c-jun. The primed cells are then able to respond to cytokines such as epidermal growth factor (EGF), tumor growth factor (TGF-&agr;), Interleukin-6 (IL-6), and hepatocyte growth factor (HGF). TGF-&agr; is synthesized by hepatocytes and acts as an autocrine factor. The in vivo response of hepatocytes to growth factors is discussed in references such as Y. Yamada et al.,
Am J Pathol
. 152:1577-89 (1998); D. E. Cressman et al.,
Science
274:1379-83 (1996); R. Taub,
FASEB J
.10:413-27 (1996); N. Fausto et al.,
FASEB J
. 9:1527-36 (1995); Webber et al.,
Hepatol
19:489-497 (1994).
Certain viruses such as hepatitis viruses show great specificity for infecting hepatocytes. Several hundred million people worldwide suffer from chronic hepatitis B virus (HBV) or hepatitis C virus infection which greatly increases their risk of developing liver cirrhosis and/or hepatocellular carcinoma (HCC). Medical therapy is generally not curative, and when available, transplanted livers can become re-infected. The only animals that can be infected with human hepatitis B virus (HBV) or human hepatitis C virus (HCV) are humans and chimpanzees, and the major tissue that is productively infected is the liver, although there have been reports of infected stromal cells.
Although in vitro models of hepatitis B and C have been used to study hepatitis virus infection (see e.g., Sureau,
Arch. Virol
.8:3-14 (1993); P. Lampertico et al.,
Hepatology
13:422-6 (1991); and N. Bishop et al.,
J Med Virol
. 31:82-9 (1990), these models are limited as to the study of disease progression. Gene expression in the in vitro models is altered from normal in vivo expression in hepatocytes. Primary hepatocyte cultures are susceptible to infection for only a few days, if at all, and do not produce the characteristic infectious particles. Human hepatitis D virus (HDV) requires envelope proteins produced by HBV, and therefore can only infect cells susceptible to HBV. The need for a good experimental system having cells that are susceptible to productive infection by viruses such as the hepatitis viruses, and other hepatic pathogens, remains.
The field of medicine relies heavily on animal models. These models provide a means of analyzing the effect of viruses and other pathogens, cytokines, environmental factors, hormones, diet, and the like. Without animal models, it is extremely difficult to perform controlled experiments. An animal model having viable human tissue provides numerous advantages over other systems such as in vitro cultured tissue. One can investigate the effect of agents on the tissue at various stages in the development of the disease. The interactions of cells, secreted age tissue can also be analyzed. A xenogeneic animal model further provides a means of testing the effect of factors and other agents on cells that are difficult to maintain in culture. Short-lived lymphocyte subsets, neural cells, complex tissues, neutrophils, etc. that cannot easily be grown in culture for extended periods of time may be examined.
In view of the many important functions performed by the liver, it is of substantial interest to develop and provide animal models comprising functional human hepatocytes that remain viable for extended periods of time. An animal model would permit investigation of the function and dysfunction of hepatocytes, the etiology of disease and the effect of pathogens and therapeutic drugs.
Many different approaches for creating an animal model for liver disease using hepatocellular transplantation have been tried over the years. Hepatocytes of the same or similar species can be stably transplanted into the liver via the spleen or portal vasculature and shown to function in a hepatocyte specific manner. While hepatocellular transplantation within the same or related species has been established, see e.g., Rhim et al.
Science
263:1149-1152 (1994), the creation of a mouse that can persistently harbor functional human hepatocytes and is susceptible to infection with HBV or HCV has not been demonstrated. Previous mouse or rat models show a low rate of persistence of hepatocyte function (K. Sanhadji et al., Bone Marrow
Trans
. 9:77-82 (1992); M. Fontaine et al.,
J. Ped. Surgery
30:56-60 (1995)). Transgenic mice expressing the hepatitis B genome replicates the virus, resulting in viremia, but not a normal course of hepatitis infection (M. J. Araki et al.,
Proc Natl Acad Sci USA
86:207-11(1989); M. B. Guidotti et al.,
J. Virol
6:6158-69 (1995)). Chimpanzees and other higher primates remain the only species besides humans susceptible to infection with hepatitis B or C viruses.
There is thus a need in the art for animal models that allow the study of human liver dysfunction, e.g., dysfunction caused by pathogenic or parasitic infection or exposure to chemical agents. There is also a need in the art for a system that allows the study of normal human liver development and function.
SUMMARY OF THE INVENTION
Non-human mammalian hosts are provided, comprising functional human hepatocytes. Isolated human hepatocytes or fragments of human hepatic tissue are introduced into the xenogeneic host in conjunction with an agent, e.g., one or more activator that stimulates signaling through the human hepatocyte growth factor receptor (hHGFR). In one embodiment, the human hepatocytes are maintained in the host by administration of one or more agent that stimulates human hepatocyte growth factor rec
Kay Mark A.
Ohashi Kazuo
Bozicevic, Field & Francis
Crouch Deborah
Field Bret E.
The Board of Trustees of the Leland Stanford Junior University
Ton Thai-An N.
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