U.S. patent application number 10/255395 was filed with the patent office on 2003-07-03 for propagation of human hepatocytes in non-human animals.
Invention is credited to Wu, Catherine H., Wu, George Y..
Application Number | 20030126626 10/255395 |
Document ID | / |
Family ID | 23713918 |
Filed Date | 2003-07-03 |
United States Patent
Application |
20030126626 |
Kind Code |
A1 |
Wu, George Y. ; et
al. |
July 3, 2003 |
Propagation of human hepatocytes in non-human animals
Abstract
The present invention relates to the preparation of non-human
animals having chimeric livers, whereby some or substantially all
of the hepatocytes present are human hepatocytes. It is based, at
least in part, on the discovery that rats, tolerized in utero
against human hepatocytes, were found to serve as long-term hosts
for human hepatocytes introduced post-natally, and the introduced
hepatocytes maintained their differentiated phenotype, as evidenced
by continued production of human albumin.
Inventors: |
Wu, George Y.; (Avon,
CT) ; Wu, Catherine H.; (Avon, CT) |
Correspondence
Address: |
BAKER & BOTTS
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
|
Family ID: |
23713918 |
Appl. No.: |
10/255395 |
Filed: |
September 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10255395 |
Sep 26, 2002 |
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09431901 |
Nov 2, 1999 |
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6525242 |
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Current U.S.
Class: |
800/8 ; 435/370;
435/456 |
Current CPC
Class: |
A61P 1/16 20180101; Y02A
50/412 20180101; A01K 2217/05 20130101; C12Y 304/21073 20130101;
C12N 9/6462 20130101; Y02A 50/388 20180101; C12N 5/067 20130101;
G01N 2333/02 20130101; A01K 67/0271 20130101; C12N 9/1247 20130101;
A61K 39/001 20130101; A61K 2035/122 20130101; Y02A 50/30 20180101;
G01N 2333/18 20130101; C12N 15/113 20130101 |
Class at
Publication: |
800/8 ; 435/456;
435/370 |
International
Class: |
A01K 067/00; C12N
005/08; C12N 015/86 |
Claims
What is claimed is:
1. A non-human animal having a liver that comprises human
hepatocytes, wherein the animal has a normal immune system but has
been rendered tolerant to human hepatocytes.
2. The non-human animal of claim 1, wherein the animal carries a
transgene that is directly toxic to hepatocytes of the non-human
animal but not specifically toxic to human hepatocytes.
3. The non-human animal of claim 2, where the transgene is a
urokinase gene operably linked to a promoter selectively active in
liver cells of the animal.
4. The non-human animal of claim 3, where the promoter is the
albumin promoter.
5. The non-human animal of claim 1, wherein the animal carries a
transgene that is indirectly toxic to hepatocytes of the non-human
animal but not specifically toxic to human hepatocytes, and wherein
the toxic effect is induced by administering an exogenous agent to
the animal.
6. The non-human animal of claim 5, where the transgene is a Herpes
simplex virus thymidine kinase gene operably linked to a promoter
selectively active in liver cells of the animal.
7. The non-human animal of claim 6, where the exogenous agent is
gancyclovir.
8. The non-human animal of claim 6, where the promoter is the
albumin promoter.
9. A method of preparing a non-human animal having a liver
comprising human hepatocytes, comprising the steps of: (i) inducing
tolerance in a non-human host animal toward hepatocytes from a
human donor and (ii) introducing hepatocytes from the human donor
into the tolerized animal produced in step (i) such that at least
some of the introduced human hepatocytes localize in the liver of
the non-human animal.
10. The method of claim 9 where tolerance is induced by
intraperitoneal injection of an antigen-containing composition
selected from the group consisting of a human cell lysate and
intact human cells.
11. The method of claim 9 where tolerance is induced by intrathymic
injection of an antigen-containing composition selected from the
group consisting of a human cell lysate and intact human cells.
12. The method of claim 9 further comprising the step of subjecting
the animal to a selection pressure which favors the proliferation
of human hepatocytes.
13. The method of claim 12 where the selection pressure is
maturation of the animal which activates the expression of a
hepatotoxic transgene in the hepatocytes of the non-human animal
host but not the donor human hepatocytes.
14. The method of claim 12 where the selection pressure is provided
by exposing the non-human animal to a toxin, wherein donor human
hepatocytes are protected against the toxin by transfection of a
transgene into the human hepatocytes, wherein the transgene
protects the donor hepatocytes from the toxic effects of the toxin,
and wherein hepatocytes of the non-human animal lack the transgene
and are therefore not protected against the toxin.
15. The method of claim 14, wherein the protective transgene
encodes a product selected from the group consisting of an
antisense RNA and a ribozyme that functionally inactivates a
cytochrome selected from the group consisting of 2E1, 1A2 and 3A4,
such that the transgene would prevent activation of acetaminophen
in human donor hepatocytes but not hepatocytes of the non-human
animal.
16. The method of claim 14, wherein the protective transgene
encodes a mutant RNA polymerase II that would render the human
donor hepatocytes resistant to phalloidin.
17. The method of claim 13 where the hepatotoxic transgene is a
urokinase gene operably linked to an albumin promoter.
18. The method of claim 12 where the selection pressure is the
administration of a compound that is metabolized to a toxic
substance in hepatocytes of the non-human animal host but not the
donor human hepatocytes.
19. The method of claim 18 where the non-human animal host carries
a Herpes simplex virus thymidine kinase gene that is selectively
expressed in hepatocytes of the animal and the administered
compound is gancyclovir.
20. A method for identifying a toxic effect of a test agent,
comprising administering the test agent to a non-human animal
having a liver that comprises human hepatocytes and subsequently
evaluating whether changes have occurred in at least one marker of
bodily function of the animal.
21. A method for identifying a toxic effect of a test agent,
comprising administering the test agent to a non-human animal
having a liver that comprises human hepatocytes and subsequently
evaluating whether changes have occurred in the viability of human
hepatocytes in the animal.
22. A model system for a human liver disease comprising a non-human
animal having a liver that comprises human hepatocytes in which an
agent associated with causation of the disease in humans has been
introduced.
23. The model system of claim 22 where the human liver disease is
alcohol-associated liver disease and where the non-human animal has
been administered an amount of alcohol effective in producing
hepatocellular degenerative changes.
24. A model system for a human liver disease caused by an
infectious agent, comprising a non-human animal having a liver that
comprises human hepatocytes infected by the infectious agent.
25. The model system of claim 24, where the infectious agent is
hepatitis C virus.
26. The model system of claim 24, where the infectious agent is
hepatitis B virus.
27. The model system of claim 24, where the infectious agent is
hepatitis A virus.
28. The model system of claim 24, where the infectious agent is
hepatitis D virus.
29. The model system of claim 24, where the infectious agent is
Yellow Fever virus.
30. The model system of claim 24, where the infectious agent is
hepatitis E virus.
31. The model system of claim 24, where the infectious agent is
malaria.
32. The model system of claim 24, where the infectious agent is
cytomegalovirus.
33. The model system of claim 24, where the infectious agent is
Epstein Barr virus.
34. A model system for a human liver disease caused by a virus,
comprising a non-human animal having a liver that comprises human
hepatocytes containing a nucleic acid of the virus, where the
nucleic acid is selected from the group consisting of a nucleic
acid comprised in the genome of the virus and a nucleic acid
transcript encoded by the genome of the virus.
35. The model system of claim 34, where the virus is hepatitis C
virus.
36. The model system of claim 34, where the virus is hepatitis B
virus.
37. The model system of claim 34, where the virus is hepatitis A
virus.
38. The model system of claim 34, where the virus is hepatitis D
virus.
39. The model system of claim 34, where the virus is Yellow Fever
virus.
40. The model system of claim 34, where the virus is hepatitis E
virus.
41. The model system of claim 34, where the virus is
cytomegalovirus.
42. The model system of claim 34, where the virus is Epstein Barr
virus.
43. A method of treating a human subject having a genetic defect,
comprising (i) propagating human hepatocytes that correct the
genetic defect in a non-human animal, where the human hepatocytes
are histocompatible with the human subject; (ii) collecting
hepatocytes from the animal; (iii) separating human hepatocytes
from non-human hepatocytes; and (iv) introducing the human
hepatocytes into the subject.
44. The method of claim 43, where the human hepatocytes are
originally obtained from the subject and then engineered to correct
the genetic defect prior to propagation in the non-human
animal.
45. A method of treating a human subject having a damaged liver,
comprising (i) propagating human hepatocytes in a non-human animal,
where the human hepatocytes are histocompatible with the human
subject; (ii) collecting hepatocytes from the animal; (iii)
separating human hepatocytes from non-human hepatocytes; and (iv)
introducing the human hepatocytes into the subject.
46. The method of claim 45, where the human hepatocytes are
originally obtained from the subject.
Description
SPECIFICATION
1. INTRODUCTION
[0001] The present invention relates to the propagation of human
hepatocytes in the livers of non-human animals that have been
tolerized to the human cells. Such animals provide an in vivo model
system of the human liver that may be used in toxicology assays and
in the study of human liver diseases, including the various forms
of hepatitis (in particular hepatitis B and C) and alcohol--induced
liver degeneration. They may also be used as a source of human
hepatocytes for reconstitution of liver tissue, thereby providing
an alternative to liver transplantation.
2. BACKGROUND OF THE INVENTION
2.1. The Need for a Cultrue System for Human Hepatocytes
[0002] To accurately study the physiology of human liver cells
(hepatocytes), scientists need a model system in which the
hepatocytes exist as they would in the intact liver. Such systems
have proven to be difficult to achieve, because when hepatocytes
are removed from their native environment, they tend to lose their
specialized functions, or "de-differentiate". The loss of
liver-specific functions makes it difficult or impossible to study
the normal functions of hepatocytes as well as their response to
chemical or biological agents. For example, research directed
toward infectious diseases of the liver, in particular viral
hepatitis, has been hampered by the lack of an adequate model
system. Hepatitis B and hepatitis C, and the problems that have
been encountered by scientists studying these infectious and
dangerous viruses, are discussed in the following subsections.
[0003] In addition, a system for propagating human hepatocytes
could be used to provide cells that could be used as an alternative
or adjunct to liver transplant. Currently, patients suffering from
liver disease may have to wait for long periods of time before a
suitable organ for transplant becomes available. After transplant,
patients need to be treated with immunosuppressive agents for the
duration of their lives in order to avoid rejection of the donor's
liver. A method for propagating the patient's own cells could
provide a source of functional liver tissue which would not require
immunosuppression to remain viable.
2.2 Hepatitis B Virus
[0004] Hepatitis B virus ("HBV") is the prototype of the
Hepadnaviridae, characterized by a unique genome structure
comprising partially double-stranded DNA (Fields Virology, 1996,
Third Edition, Fields, et al. eds., Lippincott-Raven, New York, pp.
2741-2742). In the United States, there are about a million
carriers of HBV, and the number of carriers in the world exceeds
350 million (Fields Virology, p. 2741; Petersen et al., 1998, Proc.
Natl. Acad. Sci. U.S.A. 95:310-315). In addition to causing an
acute hepatitis, viral infection may lead to chronic infection and
consequent liver failure and/or the development of hepatocellular
carcinoma (Fields Virology, pp. 2748-2751). The development of
agents that effectively treat and/or prevent the spread of the
disease has been limited by the lack of good small animal model
systems. Among the models recently developed are a transgenic mouse
model and a "Trimera", reported in Petersen et al., 1998, Proc.
Natl. Acad. Sci. U.S.A. 95:310-315 and Ilan et al., 1999,
Hepatology 29:553-562, respectively.
[0005] In the transgenic mouse model of Petersen et al., a
transgene encoding a hepatotoxic urokinase-type plasminogen
activator was introduced into RAG-2 knockout mice, which lack
mature B and T lymphocytes, and then woodchuck hepatocytes were
introduced via splenic injection. The woodchuck hepatocytes
replaced up to 90 percent of the mouse liver, and supported
woodchuck hepatitis virus (another hepadnavirus) replication
indefinitely. The replication of the virus responded to
pharmacologic agents.
[0006] In the Trimera model described by Ilan et al., nonnal mice
were preconditioned by lethal total body radiation, radioprotected
with SCID mouse bone marrow cells, and then engrafted with human
liver fragments infected ex vivo with hepatitis B.
2.3. Hepatitis C Virus
[0007] Hepatitis C virus was first characterized in 1989 (Choo et
al., 1989, Science 244: 359-362), but its existence had been
posited for many years as an elusive entity that caused flu-like
symptoms in certain patients who had received blood transfusions.
Because these symptoms were sometimes followed, years later, by
liver disease, the clinical syndrome was referred to as non A-non B
hepatitis ("NANBH").
[0008] Hepatitis C virus ("HCV") is now known to be a member of the
Flaviviridae family of viruses, which includes viruses that cause
bovine diarrhea, hog cholera, yellow fever, and tick-borne
encephalitis (Kato et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:
9524-9528; Choo et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:
2451-2455; Okamoto et al., 1991, J. Gen. Virol. 72:
2697-2704;Takamizawa et al., 1991, J. Virol. 65: 1105-1113). The
viral genome consists of an approximately 9.5 kb single-stranded,
positive-sense RNA molecule characterized by a unique open reading
frame coding for a single polyprotein (reviewed in Clarke, 1997, J.
Gen. Virol 78: 2397-2410 and Major and Feinstone, 1997, Hepatology
25: 1527-1538). Based upon phylogenetic analysis of the core, EI,
and NS5 regions, HCV has been found to be genetically
heterogeneous, with at least six genotypes and more than 30
subtypes dispersed throughout the world (Major and Feinstone, 1997,
Hepatology 25: 1527-1538; Clarke, 1997, J. Genl. Virol 78:
2397-2410).
[0009] HCV has been estimated to infect 170 million people
worldwide, which is more than four times the number of persons
infected with human immunodeficiency virus ("HIV"), and the number
of HCV-associated deaths may eventually overtake deaths caused by
AIDS (Cohen, 1999, Science 285: 26-30). The Center for Disease
Control has calculated that HCV may be harbored by 1.8 percent of
the U.S. population. (Id.). The only available therapy is
interferon, but most HCV isolates are resistant (Thomas et al.,
1999, Hepatology 29: 1333), although more promising results were
obtained when interferon was combined with ribavirin (Cohen et al.,
1999, Science 285: 26-30 citing Poynard et al., 1998, Lancet
352:1426-1432 and Davis et al., 1998, N. Engl. J. Med.
339:1493-1499). Unfortunately, the interferon/ribavirin combination
is less effective against the most common HCV genotype found in the
U.S., with only 28 percent of persons infected with that genotype
exhibiting a sustained response to treatment. (Davis et al., 1998,
N. Engl. J. Med. 339:1493-1499).
[0010] The development of more successful forms of therapy (and our
understanding of HCV biology) has been hampered by the absence of a
good model system for HCV infection. Only humans and certain higher
primates are susceptible to infection (Feinstone et al., 1981, J.
Infect. Dis. 144: 588). A variety of mammalian cell systems which
support the growth of HCV have been reported which rely on the use
of strand-specific RT-PCR as evidence of virus replication (Major
and Feinstone, 1997, Hepatology 25:1527-1538 citing Mitzutani et
al., 1995, Biochem. Biophys. Res. Commun. 212: 906-911; Shimizu and
Yoshikura, 1994, 68: 8406-8408; Kato et al., 1995, Biochem.
Biophys. Res. Commun. 206: 863-869; Cribier et al., 1995, J. Gen.
Virol. 76: 2485-2491; and Yoo et al., 1995, J. Virol. 69:
32-38).
[0011] As reviewed in Clarke (supra), there have been reports of
viral replication in systems based on hepatic tissue (Ito, et al.,
1996, J. Gen. Virol. 77: 1043-1054), peripheral blood mononuclear
cells (Willems et al., 1996, J. Med. Virol. 42: 272-278; Zignego et
al., 1992, J. Hepatology 15: 382-386), human T and B cell lines
(Bertolini et al., 1993, Res. Virol 144: 281-285; Shimizu et al.,
1992, Proc. Natl. Acad. Sci. U.S.A. 89: 5477-5481), human fetal
liver cells (Iacovacci et al., 1993, Res. Virol. 144: 275-279),
chimpanzee hepatocytes (Lanford et al., 1994, Virol. 202: 606-614),
Daudi B-cells (Nakajima et al., 1996, J. Virol. 70: 3325-3329), and
the human T cell leukemia virus type I-infected T cell line MT-Z
(Mitzutani et al., 1995, Biochem. Biophys. Res. Comm. 212: 906-911;
Sugiyama et al., 1997, J. Gen. Virol. 78: 329-336). None of these
systems has, however, proved satisfactory.
[0012] Hepatitis C infected human liver tissue was transplanted
into Trimera mice described in the preceding section, as reported
by Galun et al., 1995, J. Infect. Dis. 172:25-30.
[0013] A newer system was recently reported by Lohmann et al.
(1999, Science 285: 110-113) in which subgenomic HCV RNA replicons
were transfected into a human hepatoma cell line and found to
replicate to high levels. Nonetheless, this system does not
generate virus and therefore is not a model of productive infection
(Cohen, supra).
2.3. Hepatocytes for Liver Reconstitution
[0014] Reconstitution of liver tissue in a patient by the
introduction of hepatocytes (also referred to as "hepatocyte
transplantation") is a potential therapeutic option for patients
with acute liver failure, either as a temporary treatment in
anticipation of liver transplant or as a definitive treatment for
patients with isolated metabolic deficiencies (Bumgardner et al.,
1998, Transplantation 65: 53-61). Animal models have been developed
for studying the effectiveness of hepatocyte transplantation in the
context of pharmacologically or surgically induced liver failure
(Id, citing Mito et al., 1993, Transplant Rev. 7: 35. Takeshita et
al. 1993, Cell Transplant 2: 319; Sutherland et al., 1977, Surgery
82: 124; Sommer et al., 1979, Transplant Proc. 9: 578; and
Demetriou et al., 1988, Hepatology 8: 1006), or for the treatment
of isolated errors of metabolism (Wiederkehr et al., 1990,
Transplant 50: 466; Onodera et al., 1995, Cell Transpl. 4 (Supp.
1): 541; Cobourn et al., 1987, Transpl. Proc. 19: 1002; Rozga et
al., 1995, Cell Transplant 4: 237; Kay et al., 1994, Hepatology 20:
253; Matas et al., 1976, Science 192: 892; Holzman et al., 1993,
Transplantation 55: 1213; Moscioni et al., 1989, Gastroenterol. 96:
1546; Groth et al., 1977, Transplant Proc. 9: 313). Use of
transfected hepatocytes in gene therapy of a patient suffering from
familial hypercholesterolemia has been reported in Grossman et al.,
1994, Nat. Genet. 6: 335.
[0015] A major obstacle to achieving therapeutic liver
reconstitution is immune rejection of transplanted hepatocytes by
the host, a phenomenon referred to (where the host and donor cells
are genetically and phenotypically different) as "allograft
rejection". Immunosuppressive agents have been only partially
successful in preventing allograft rejection (Javregui et al.,
1996, Cell Transplantation 5: 353-367, citing Darby et al., 1986,
Br. J. Exp. Pathol. 67: 329-339; Maganto et al., 1988, Eur. Surg.
Res. 20: 248-253; Makowka et al., 1986, Transplantation 42:
537-541). The three main alternative approaches which have been
explored are 1) physically shielding transplanted cells from the
host immune system, for example, in an alginate-polylysine or
chitosan capsule; 2) depletion of antigen presenting cells; or 3)
induction of alloantigen-specific tolerance in the host (Javregui
et al., supra). Chowdhury has tested the hypothesis that
intrathymic injection of donor rat splenocytes may result in
suppression of allograft hepatocyte rejection in peripheral
lymphocyte depleted adult rats (Jauregui et al., supra, citing
Fabrega et al., 1995, Transplantation 59: 1362-1364). In that study
long-term tolerization occurred with administration of splenocytes
but not hepatocytes.
[0016] For successful reconstitution, the age of the donor cells
has been considered significant. Cusick et al. (1997, J. Ped. Surg.
32: 357-360) report that transplanted fetal hepatocytes had a
significant survival advantage over adult hepatocytes, independent
of recipient age. However, Rhim et al. (1994, Science 263:
1149-1152) demonstrated that adult mouse liver cells could
proliferate when introduced into the livers of congenic transgenic
mice carrying a hepatotoxic transgene (urokinase under the control
of the albumin promoter, which is liver-specific and only active
postnatally). The donor cells were observed to have divided at
least 12 times (reconstitution of an entire liver from one
hepatocyte would require 28 cell doublings).
3. SUMMARY OF THE INVENTION
[0017] The present invention relates to the preparation of
tolerized non-human animals having chimeric livers, wherein some or
a majority of the hepatocytes present are human hepatocytes. It is
based, at least in part, on the discovery that rats, tolerized in
utero against human hepatocytes, were found to serve as long-term
hosts for human hepatocytes introduced postnatally, and that the
introduced hepatocytes maintained their differentiated phenotype,
as evidenced by continued production of human albumin.
[0018] In a first embodiment, the present invention provides for a
method of preparing a non-human animal having a liver comprising
human hepatocytes, comprising (i) inducing tolerance in an
immunocompetent host non-human animal, where the animal is
preferably a fetus or a neonate; and (ii) introducing human
hepatocytes into the tolerized animal, preferably postnatally and
preferably by intra-splenic injection. In specific non-limiting
embodiments, the host animal is subjected to a selection pressure
which favors survival and/or proliferation of human, rather than
host animal, hepatocytes.
[0019] In a second embodiment of the invention, an animal having a
chimeric liver, prepared as described above, may be used as a model
system for human hepatocyte function in a toxicology study. Because
the human hepatocytes maintain their differentiated state and are
situated in their natural anatomic location, this model system
recapitulates the metabolic fate of test agents as they pass from
the site of administration through the liver.
[0020] In a third embodiment of the invention, an animal having a
chimeric liver may be used as a model system for human liver
disease. Such model systems are particularly useful for diseases
which specifically effect human (or primate), but not non-human (or
non-primate) livers, such as hepatitis B and hepatitis C infection
and alcohol-induced liver degeneration/fibrosis. Immunocompetent
chimeric animals of the invention exhibit the further advantage of
having an immune system which is intact but for exhibiting
tolerance toward the human cells comprised in the animal's
liver.
[0021] In a fourth embodiment of the invention, an animal having a
chimeric liver may be used as a source of human hepatocytes which
may be used therapeutically. As non-limiting examples, such human
hepatocytes may be used in gene therapy applications or to
reconstitute liver tissue in a human host whose own liver has been
substantially damaged. Large animals having a chimeric liver may be
particularly desirable for such embodiments.
4. DESCRIPTION OF THE FIGURES
[0022] FIG. 1. .sup.3[H]-thymidine incorporation in mixed
lymphocyte assays where the responder cells were rat spleen cells
and the stimulator cells were irradiated human hepatocytes. "Spleen
(iu)" designates spleen cells from rats tolerized by intrauterine
injection of human hepatocyte lysates, and "spleen (iu/is)"
designates spleen cells from rats tolerized as fetuses with human
hepatocyte lysates followed by intrasplenic transplantation of
human hepatocytes after birth.
[0023] FIG. 2. Western blot, using anti-human albumin antibody as a
probe, of human serum albumin (lane 1), serum from a tolerized rat
six weeks after intrasplenic injection (lane 2), and sera from a
tolerized rat, injected with human hepatocytes, 24 hours (lane 3),
and eight days (lane 4) after a second injection of human
hepatocytes. Lane 5 contains sernm from a non-tolerized rat eight
days after a second injection of human hepatocytes.
[0024] FIG. 3. Western blot, using anti-human albumin antibody as a
probe, of human serum albumin (lane 1), rat serum albumin (lane 2),
and serum from a tolerized rat that had received an intrasplenic
injection of human hepatocytes, one week (lane 3), two weeks (lane
4) and three weeks (lane 5) after injection with human
hepatocytes.
[0025] FIGS. 4A-D. Immunofluorescence studies using anti-human
albumin as primary antibody and fluorescent Texas red--coupled
secondary antibody. (A) Anti-human albumin antibody binding to
control (non-chimeric) rat liver; (B) anti-human albumin antibody
binding to chimeric rat liver three weeks after injection with
human hepatocytes; (C) same as B, without secondary antibody
visualization; and (D) anti-human albumin antibody binding to the
liver of a rat that had been tolerized with a human hepatocyte
lysate but did not receive subsequent injection of viable liver
cells.
[0026] FIG. 5. The same section of chimeric rat liver shown in FIG.
4B to express human albumin, stained with hematoxylin and eosin to
demonstrate normal histology.
[0027] FIGS. 6A-D. Immunofluorescence studies using primary and
secondary antibodies as in FIGS. 4A-D, showing (A) a section of
liver from a tolerized rat six weeks after intrasplenic injection
with human hepatocytes, stained with both antibodies; (B) as in
(A), but without secondary antibody staining; (C) as in (A), but
with no antibody binding; and (D) a section of liver from a
non-tolerized rat, six weeks after intrasplenic injection of human
hepatocytes, stained with both antibodies.
[0028] FIG. 7. Western blot, using anti-human albumin antibody as a
probe, of human serum albumin (lane 1), rat serum (lane 2) and sera
from a chimeric rat which had been tolerized by intrathymic
injection of human hepatocytes, at varius times after intrasplenic
injection with human hepatocytes (lane 3=2 days, lane 4=2 weeks,
lane 5=3 weeks, lane 6 weeks, lane 7=6 weeks).
[0029] FIGS. 8A-F. Immunofluorescence studies of liver sections
from tolerized rats injected with human hepatocytes and inoculated
with hepatitis B virus (HBV) at 1 week, 6 weeks, and 14 weeks
following inoculation, stained with anti-albumin primary and Texas
red conjugated secondary antibody (FIGS. 8A, 8C and 8E, for weeks
1, 6 and 14, respectively) or anti-hepatitis B surface antigen
(HBsAg) antibody and FITC-conjugated secondary antibody (FIGS. 8B,
8D, and 8F for weeks 1, 6 and 14, respectively).
[0030] FIGS. 9A-H. Immunofluorescence studies of liver sections
from rats that were either (i) tolerized, injected with human
hepatocytes, and inoculated with HBV (CA2)(FIGS. 9A and 9B); (ii)
tolerized and injected with human hepatocytes but not inoculated
(CA3) (FIGS. 9C and 9D); (iii) tolerized and inoculated with HBV,
without injection of human hepatocytes (CA5) (FIGS. 9E and 9F); or
tolerized, injected with human hepatocytes and inoculated with HBV
(CA2) but not reacted with primary anti-albumin or anti-HBsAg
antibodies (FIGS. 9G and 9H). Sections were stained with
anti-albumin primary and Texas red conjugated secondary antibody
(FIGS. 9A, 9C, 9E, and 9G) or anti-HBsAg antibody and
FITC-conjugated secondary antibody (FIGS. 9B, 9D, 9F, and 9H).
[0031] FIG. 10. Photograph of an ethidium bromide stained gel of
products of RT-PCR of human albumin mRNA, the lanes containing the
RT-PCR products resulting from experiments using, as template, RNA
from: lane 2=rat; lane 3=human; lane 4=HepG2.2.15; lane 5=Rat CA1
(tolerized, injected with human hepatocytes, subsequently
inoculated with HBV); lane 6=Rat CA2 (tolerized, injected with
human hepatocytes, subsequently inoculated with HBV); lane 7=rat
CA3 (tolerized and injected with human hepatocytes but not
inoculated with HBV); lane 8=rat CA4 (tolerized and injected with
human hepatocytes but not inoculated with HBV); lane 9=(tolerized
and inoculated with HBV, without injection of human hepatocytes);
lane 10=(tolerized and inoculated with HBV, without injection of
human hepatocytes); lane 11=rat CA7 (treated with saline, negative
control); and where lane 1=1,000 bp ladder.
[0032] FIGS. 11A-B. Photograph of an ethidium bromide stained gel
of products of RT-PCR of human albumin mRNA (FIG. 11A) and HBV RNA
(FIG. 11B), the lanes containing the RT-PCR products resulting from
experiments using, as template, RNA from : lane 2=rat; lane
3=human; lane 4=HepG22.2.15; lane 5=Rat CA2 (tolerized, injected
with human hepatocytes, inoculated with HBV) 1 week
post-inoculation; lane 6=Rat CA2 6 weeks post-inoculation, lane
7=rat CA2 14 weeks post-inoculation, where lane 1=1,000 bp
ladder.
[0033] FIG. 12. Photograph of an ethidium bromide stained gel of
products of RT-PCR of human hepatitis B viral RNA, the lanes
containing RT-PCR products resulting from experiments using, as
template, RNA from: lane 2=rat; lane 3=human; lane 4=HepG22.2.15;
lane 5=Rat CAl (tolerized, injected with human hepatocytes and
inoculated with HBV); lane 6=Rat CA2 (tolerized, injected with
human hepatocytes, inoculated with HBV); lane 7=Rat CA3 (tolerized
with human hepatocytes but not inoculated with HBV); lane 8=Rat CA4
(tolerized with human hepatocytes but not inoculated with HBV);
lane 9=Rat CA5 (tolerized with human hepatocytes, not injected with
human hepatocytes, inoculated with HBV); lane 10=Rat CA6 (tolerized
with human hepatocytes, not injected with human hepatocytes,
inoculated with HBV); where lane 1=1,000 bp ladder.
[0034] FIGS. 13A-C. Photomicrographs of hematoxylin-eosin stained
liver sections, at low (20.times.) magnification, of liver sections
from a rat tolerized, transplanted with human hepaotcytes, and
inoculated with HBV, (13A) 1 week, (13B) 6 weeks, or (13C) 14 weeks
post-inoculation.
[0035] FIGS. 14A-C. Photomicrographs of hematoxylin-eosin stained
liver sections, at high (40.times.) magnification, of liver
sections from a rat tolerized, transplanted with human hepaotcytes,
and inoculated with HBV, (13A) 1 week, (13B) 6 weeks, or (13C) 14
weeks post-inoculation.
5. DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention relates to tolerized non-human animals
having chimeric livers comprising human hepatocytes, methods for
preparing such animals, and the use of such animals either as model
systems for assaying toxicology or studying human liver disease or
as sources of human hepatocytes for re-introduction into a human
host. For purposes of clarity, the description of the invention is
presented as the following subsections:
[0037] i) producing animals having chimeric livers;
[0038] ii) toxicology model systems;
[0039] iii) model systems for liver diseases; and
[0040] iv) chimeric animals as a source of hepatocytes for liver
reconstitution.
[0041] The subject animals of the invention are referred to herein
alternatively as "non-human animals having chimeric livers" or
simply "chimeric animals". Both these terms are defined as
tolerized non-human animals having livers which comprise human
hepatocytes. In addition to the human hepatocytes, the livers of
the chimeric animals may also include hepatocytes and
non-hepatocyte elements (e.g., biliary and vascular endothelial
cells, Kupffer cells, etc.) endogenous to the animal itself. Human
cell types other than hepatocytes may also be present. Preferably,
the percentage of human hepatocytes (relative to the total number
of hepatocytes present) is at least 10 percent, more preferably at
least 20 percent, or at least 50 percent, or at least 80
percent.
[0042] In particular, chimeric animals are created by introducing
human hepatocytes (and possibly additional cell types) into an
animal rendered immunologically tolerant to the introduced human
cells. As such, the animals may be referred to as being "hosts" to
the human cells, where a human being that is a source of such cells
may be referred to as a "donor". The term "tolerant", as used
herein, does not refer to a state of general immunosuppression (as
might be achieved, for example, by treatment with cyclosporine, or
as may exist in an animal with a generalized B cell and/or T cell
deficiency) but rather indicates a state of antigen-induced
non-responsiveness of lymphocytes achieved by clonal deletion,
cell-mediated suppression, or anergy (see, for example, Davies,
1997, "Introductory Immunobiology", Chapman & Hall, London, p.
366) directed specifically toward the introduced human cells.
5.1. Producing Animals Having Chimeric Livers
[0043] The present invention provides for a method of preparing a
non-human animal having a liver comprising human hepatocytes,
comprising (i) inducing tolerance in a host animal, where the
animal is preferably a fetus or a neonate; and (ii) introducing
human hepatocytes into the tolerized animal, preferably postnatally
and preferably by intra-splenic injection. In specific embodiments,
the host animal is subjected to a selection pressure which favors
survival and/or proliferation of human, rather than host animal,
hepatocytes. A detailed non-limiting description of these features
of the invention is set forth in the following subsections.
5.1.1. Host Animals
[0044] Non-human animals which may serve as hosts according to the
invention are preferably mammals, and include, but are not limited
to, mice, hamsters, rats, rabbits, dogs, goats, sheep, pigs,
cattle, etc.. In particular non-limiting embodiments of the
invention, the host animal is a transgenic animal carrying, as a
transgene, a gene which, when expressed in hepatocytes, is directly
or indirectly (i.e. via a metabolite) toxic to those cells.
Examples of such genes are the urokinase gene which is directly
toxic (Sandgren et al., 1991, Cell 66:245), and the Herpes simplex
virus ("HSV") thymidine kinase gene ("HSV-TK"); which converts the
drug gancyclovir into a toxic form and is therefore indirectly
toxic (Smythe et al., 1995, Ann. Surg. 222:78-86). Preferably, the
gene is operably linked to a promoter which is selectively active
in hepatocytes, such as the albumin promoter, the PEPCK promoter,
and the hepatitis B surface antigen promoter. To avoid destroying
the animal's liver prior to colonization with human hepatocytes, it
is desirable to utilize a promoter that is not particularly active
pre-natally. Otherwise, such transgenic animals may die in utero.
Other promoters inducible by agents that could be locally
administered into the liver may also be suitable, such as the
metallothionein promoter (which is inducible by heavy metal ions;
Palmiter et al., 1982, Cell 29:701). Such genes are not
specifically toxic to human hepatocytes, although there may be some
"bystander effect" whereby a limited number of the human
hepatocytes are killed.
[0045] In one specific, non-limiting embodiment of the invention,
transgenic mice carrying an albumin promoter/urokinase transgene
may be used as hosts. Urokinase is a plasminogen activator that is
useful clinically in dissolving blood clots. When introduced into
hepatocytes by an adenoviral vector, it was shown to be toxic to
those cells (Lieber et al., 1995, Proc. Natl. Acad. Sci. U.S.A.
92:6210-6214). In addition, Sandgren et al. prepared a transgenic
mouse containing the mouse urokinase gene driven by a mouse albumin
enliancer/promoter (Sandgren et al., 1991, Cell 66:245-256).
Because albumin is not produced by the fetal liver (Krumlaufet al.,
1985, Cold Spring Harbor Symp. Quant. Biol. 5-0:371-378), animals
survived in utero because urokinase was not produced. However,
after birth, with activation of the albumin promoter, the liver was
destroyed due to the presence of urokinase. To produce such a
transgenic mouse for use as a host, heterozygote transgenic mice,
B6SJL background, may be obtained from Jackson Laboratories, Stock
No. 002214, which contain the mouse urokinase gene driven by a 3.5
kb mouse albumin promoter sequence with a human growth hormone poly
A addition site. Pregnant mice from heterozygotic matings may be
used to generate homozygous offspring. The number of copies of the
urokinase transgene present in each animal at birth may be
determined from DNA extracts of tail snips, where the DNA may be
digested with Kpn 1, which cuts once within the urokinase gene, and
Southern blotting using a detectably labeled probe specific for the
urokinase gene, such as 5'-TGTGCTTATG TAGCCATCCA GCGAGTCCCC-3' (SEQ
ID NO: 1). Because somatic mutations leading to inactivation of the
urokinase gene may occur, it may be desirable to use breeding pairs
of male and female mice successfully rescued into adulthood by
introduction of human hepatocytes to generate litters of homozygous
offspring. Further, in previous studies on mice carrying a
urokinase transgene, inactivating mutations in the urokinase gene
were found to result in proliferation of those cells with that
somatic mutation while the homozygous cells failed to grow. The
proliferating cells, as expected, had higher ploidy than those less
actively proliferating (Sandgren et al., 1991, Cell 66:245-256).
Thus, the copy number of human DNA, if measured during
proliferation of human hepatocytes may be biased, and not reflect
the number of cells due to polyploidy. For this reason, the number
of human cells may be better estimated by measuring markers
specific for human hepatocytes, such as, but not limited to, the
human albumin gene or its protein product.
[0046] In another specific non-limiting embodiment of the
invention, transgenic mice carrying an albumin
promoter/HSV-thymidine kinase gene may be used as hosts. Thymidine
kinase of HSV differs from mammalian thymidine kinases in its
ability to phosphorylate the drug gancyclovir (Fyfe et al., 1978,
J. Biol. Chem. 253:8721-8727). In so doing, it converts the
non-toxic agent into a toxic form (De Clerq, 1984, Biochem.
Biopharmacol. 33:2159-2169). In a specific non-limiting embodiment,
the HSV-TK gene (as present in plasmid pLTR-DTK, as developed by D.
Klatzmann, Universit Pierre et Marie Curie, Paris, France) may be
linked to an albumin promoter prepared by excising a 3.2 kb
fragment of the mouse albumin promoter (for example from
palb.sub.9-12LDLR, James Wilson, University of Pennsylvania,
Philadelphia, Pa.) using Bgl II and Sal 1 restriction enzymes
(Wilson et al., 1992, J. Biol. Chem. 267:963-967), and placing the
promoter fragment in a polylinker site immediately upstream of the
HSV-TK gene. Using this plasmid, founder outbred CD1 mice may be
prepared and mated to normal CD1 mice to generate heterozygotes,
detected by DNA analysis of tail snips using an HSV-TK specific
detectably labeled probe. A breeding pair of heterozygotes may then
be used to produce mice homozygous for the albumin promoter/HSV-TK
transgene. It should be noted that the natural HSV-TK gene contains
elements that activate the gene in the testes, which may result in
sterile animals that cannot be used as breeders. Accordingly, a
version of the gene which lacks these elements is preferred, such
as the gene contained in plasmid pLTR-.DELTA.TK (all such variant
genes, as well as the wild-type, are considered HSV-TK genes).
Breeding of transgenic mice with this specific construct confirmed
the success of the deletion (Salomon et al., 1995, Mol. Cell. Biol.
15:5322-5328). Further, a gancyclovir dose-related (Culver et al.,
1992, Science 256:1550-1552) bystander effect of the HSV-TK gene
product has been observed whereby nearby cells lacking the
transgene are destroyed (Kolberg, 1994, J. NIH Res. 6:62-64).
Accordingly, it may be desirable to evaluate different doses of
gancyclovir and identify the minimum dose required to produce
maximal human hepatocyte proliferation.
5.1.2. Tolerization
[0047] Non-human animals which are to be used as hosts for human
hepatocytes may be rendered tolerant to those hepatocytes by
administration of the relevant antigen(s), preferably in the
context of human cells or a lysate prepared from human cells, more
preferably using human cells from the same individual who is to
serve as the hepatocyte donor. Tolerizing antigen(s) may be
administered as whole cells, a cell extract or one or more purified
component thereof. The source of tolerizing antigen(s) may be
hepatocytes, but may alternatively be cells of another type, or a
mixture of different types of cells. For example, cells prepared
from a specimen of human liver tissue may be used as a source of
tolerizing antigen(s); such cells may include not only hepatocytes
but also fibroblasts, cells of the biliary system, vascular
endothelial cells, Kupffer cells, etc.. As another example, human
splenocytes or lysates thereof may be used to induce tolerance.
Cells to be used in tolerization are preferably cleared of
undesirable constituents. For example, if the animal is eventually
to be used as a model system for a disease where an immune response
to an infectious agent is desirably left intact, the animal should
not be tolerized against the infectious agent. Alternatively, if
the animal is to be used as a host to support the proliferation of
human hepatocytes to be used to reconstitute the liver of a person
having liver damage caused by an infectious agent, it is desirable
not to tolerize the host animal toward the infectious agent or to
introduce the infectious agent into the host animal at any time.
The cells or lysate are introduced in a physiologically compatible
solution; herein, volumes administered refer to cells or lysate
comprised in such a solution.
[0048] While the host animal may potentially be of any age when
tolerized, tolerization is likely to become more difficult as age
of the animal increases. Preferably, the animal is still an infant
when tolerized; more preferably, the animal is a neonate, or
tolerized in utero. If the intended host animal is a rat, the
preferable upper age limit for tolerization is 18 days
post-conception (in utero), and the more preferable age for
tolerization is 17 days post-conception (in utero), or within 24
hours after birth. If the intended host animal is a mouse, the
preferable upper age limit for tolerization is 18 days
post-conception (in utero), and the more preferable age for
tolerization is 17 days post-conception (in utero), or within 24
hours after birth. If the intended host animal is a pig, the
preferable upper age limit for tolerization is 90 days
post-conception, and the more preferable age for tolerization is 80
days post-conception, when the animal is still in utero, or within
24 hours after birth.
[0049] Tolerization may be accomplished by any route, including but
not limited to intravenous, intraperitoneal, subcutaneous, and
intrathymic routes. Preferred methods of tolerization include
inoculation of human cells into the thymus or
intraperitoneally.
[0050] As a specific, non-limiting example, where the intended host
animal is a rat, tolerance may be induced by inoculating lysate
prepared from 1.times.10.sup.4-1.times.10.sup.6 and preferably
0.5.times.10.sup.5 human hepatocytes into the peritoneum of a 15-18
day old, and preferably a 17 day old, rat fetus in utero under
transillumination. The lysate may be prepared by sonicating a
suspension of the appropriate number of human hepatocytes. The same
numbers of whole cells may also be inoculated into the peritoneum
during the aforesaid time periods. If the intended host animal is a
mouse, the number of human hepatocytes represented in the lysate
may be 1.times.10.sup.3-1.times.10.sup.5 and preferably 10.sup.4
and intraperitoneal inoculation may be performed between days 15
and 18 post conception. If the intended host animal is a pig, the
number of human hepatocytes represented in the lysate may be
between about 10.sup.5 and 10.sup.6 or the same number of whole
cells and intraperitoneal inoculation may be performed at between
about 75 and 90 days post-conception. Alternatively,
intraperitoneal inoculation can be performed while the animals are
neonates.
[0051] As a second non-limiting example, tolerance may be induced
by intrathymic injection according to a method as described in
Fabrega et al., 1995, Transplantation 59:1362-1364. Either whole
cells or a cell lysate may be administered. In particular, where
the intended host animal is a rat, about
1.times.10.sup.2-1.times.10.sup.4, preferably 100, human
hepatocytes (or a lysate thereof) in between about 1 and 10
microliters, preferably about 5 microliters, may be injected into
the thymus of a newborn (neonatal) rat, preferably within 1-2 hours
of birth. Where the intended host animal is a mouse, about
1.times.10.sup.2-1.times.10.sup.4 and preferably 100 human
hepatocytes (or a lysate thereof) in between about 1 and 10
microliters and preferably about 5 microliters may be injected into
the thymus of a mouse that is up to 3 months old and preferably a
neonate, e.g. within 1-2 hours or within 24 hours of birth. Where
the intended host animal is a pig, about 10.sup.5-10.sup.6 human
hepatocytes (or a lysate thereof) in between about 50 and 200
microliters may be injected into the thymus of an infant pig that
is preferably up to one week old. As a specific example, a neonatal
mouse may be anesthetized by chilling on ice, the thoracic area may
be cleaned with alcohol and betadine swipes, the thymus may be
visualized through the translucent skin of the newborn, and a 1-2
mm incision may be made with ophthalmic scissors to expose the
thymus. The human cells or human cell lysate may then be slowly
injected into the thymus, and then the incision may be closed with
a sterile nylon suture. The incision area may then be recleaned and
the mouse placed on a warming pad and returned to its mother as
soon as possible.
[0052] The success of tolerization may be assessed by proceeding to
introduce human hepatocytes into the animal, and determine whether
or not they survive long-term (for example, by monitoring the
production of human serum albumin; see infra). Alternatively, the
ability of lymphocytes from the animal to react with donor human
hepatocytes may be evaluated using standard immunologic techniques,
such as methods that determine T cell proliferation in response to
donor hepatocytes, the induction of a cytotoxic T cell response, or
mixed lymphocyte reaction.
5.1.3. Introduction of Human Liver Cells
[0053] Human liver cells may then be introduced into host animals
rendered tolerant as set forth in the preceding section. The
hepatocytes may preferably be introduced via intrasplenic
injection, although other routes may also be used, such as direct
injection into the liver parenchyma, under the liver capsule, or
via the portal vein.
[0054] As a specific non-limiting example, where the intended host
animal is a rat tolerized as set forth above, between about
10.sup.6-5.times.10.sup.7 human hepatocytes, preferably about
2.times.10.sup.6 hepatocytes, may be introduced into a tolerized
rat within about 24 hours after birth by anesthetizing the animal,
making a 3-4 mm incision in the left paracostal area to visualize
the spleen (Marucci et al., 1997, Hepatol. 26:1195-1202), and
injecting the donor cells in a volume of approximately about 50-300
microliters, and preferably about 200 microliters, of sterile
medium. Where the intended host animal is a tolerized mouse, the
number of human hepatocytes introduced by an analogous procedure
may be between about 5.times.10.sup.3 and 5.times.10.sup.6,
preferably about 10.sup.5 in a volume of about 25-200 microliters,
and preferably about 100 microliters, of sterile medium, and the
human hepatocytes are administered between about one day and two
months, preferably 3-4 days, after tolerization. Where the intended
host animal is a tolerized pig, the number of human hepatocytes may
be between about 10.sup.8-10.sup.10, preferably about 10.sup.9, in
a volume of about 10-20 milliliters of sterile medium and the human
hepatocytes are administered about one and seven days after birth
or about 35 days after tolerization.
[0055] Human hepatocytes may be obtained from a commercial source,
for example, Clonetics Corporation, 8830 Biggs Ford Road,
Walkersville, Md. 21793, which sells normal human hepatocytes as
catalog number CC-2591.
[0056] Alternatively, human hepatocytes may be prepared from a
donor as follows. The source of cells may be from a liver biopsy
taken percutaneously or via abdominal surgery, or from liver tissue
obtained postmortem. The source of cells should be maintained in a
manner which protects cell viability. In one specific non-limiting
embodiment, human hepatocytes may be prepared using the technique
described in Guguen-Guillouzo et al., 1982, "High yield preparation
of isolated human adult hepatocytes by enzymatic perfusion of the
liver", Cell Biol. Int. Rep. 6:625-628. Briefly, the method of
Guguen-Guillouzo et al. involves (i) isolating a liver or a portion
thereof from which hepatocytes are to be harvested; (ii)
introducing a cannula into the portal vein or a portal branch;
(iii) perfusing the liver tissue, via the canula, with a
calcium-free buffer followed by an enzymatic solution containing
0.025% collagenase (e.g., Type 4, from Sigma Chemical Company) in
0.075% calcium chloride solution in HEPES buffer at a flow rate of
between 30 and 70 milliliters per minute at 37.degree. C.; then
(iv) mincing the perfused liver tissue into small (e.g. about 1
cubic millimeter) pieces; (v) continuing the enzymatic digestion in
the same buffer as used in step (iii) for about 10-20 minutes with
gentle stirring at 37.degree. C. to produce a cell suspension; and
(iv) collecting the released hepatocytes by filtering the cell
suspension produced in step (v) through a 60-80 micrometer nylon
mesh. The collected hepatocytes may then be washed three times in
cold HEPES buffer at pH 7.0 using slow centrifugation (e.g.,
50.times.g for five minutes) to remove collagenase and cell debris.
Non-parenchymal cells may be removed by metrizamide gradient
centrifugation. If the amount of liver tissue is too small to
perform the above perfusion procedure, for example, less than 100 g
of tissue, then the tissue may be minced and digested with
collagenase solution with gentle stirring and processed according
to steps (iv) and (v) of this paragraph.
[0057] It may be desirable to separate human hepatocytes prepared
as set forth above into a subset for introduction into animals and
another subset which is undesirable to propagate. For example, if a
human subject is to serve as a donor for hepatocytes which are to
be propagated in a chimeric animal according to the invention and
then reintroduced into the subject, e.g., to reconstitute a liver
damaged by infectious disease or malignancy, it would be desirable
not to propagate hepatocytes which are infected or which have
undergone malignant transformation. In such a situation, it would
be desirable to eliminate infected or malignant hepatocytes from
the population of hepatocytes which is to be introduced into the
host animal. Elimination of unwanted cells can be performed by
standard cell sorting techniques, for example fluorescence
activated cell sorting using an antibody specific for the
infectious agent or for malignant transformation. Alternatively,
undesirable cells may be eliminated or attenuated by treatment with
antiviral or antimicrobial compounds, radiation, antibody-ligated
toxins, culture techniques, etc..
5.1.4. Favoring Proliferation of Human Hepatocytes
[0058] In particular non-limiting embodiments of the invention,
selection pressure may be used to favor the proliferation of human
hepatocytes. Such selection pressure is defined herein as including
any condition, preexisting in the host animal at the time of
introduction of donor cells or imposed thereafter, which results in
a greater likelihood that human hepatocytes, rather than host
hepatocytes, will proliferate.
[0059] For example, the selection pressure may result from the
presence of a trausgene that decreases the viability of host
hepatocytes, either intrinsically (directly) or by administration
of an activating agent (indirectly). Alternatively, human donor
hepatocytes can be transfected with a protective gene that will
enable those cells to survive subsequent exposure to a hepatotoxin.
In one specific non-limiting example, the transgene may be the
albumin promoter/urokinase construct, whereby as the host animal
matures and the albumin promoter becomes active, host hepatocytes
may be eliminated by the toxic effects of urokinase. In such cases,
the selection pressure is maturation of the animal with consequent
transgene activation. In a second specific non-limiting example,
the transgene may be the albumin promoter/HSV-TK construct, whereby
when gancyclovir is administered to the host animal (e.g., as an
intraperitoneal injection of 250 mg/kg gancyclovir in sterile PBS),
hepatocytes of the transgenic host may be selectively killed. In
such embodiments, the death of host hepatocytes would be expected
to favor compensatory proliferation of human hepatocytes. This can
occur because of the known property of parenchymal liver cells to
proliferate during conditions that stimulate regeneration.
[0060] It may be preferable to effect stepwise attenuation of host
hepatocytes rather than eliminate a majority in a short period of
time, as the sudden loss of liver function could result in death of
the animal and/or conditions that would disfavor the establishment
of a human hepatocyte population in the host liver. For example,
administration of several doses of gancyclovir to a host animal
transgenic for the albumin promoter/HSV-TK construct, beginning
before and continuing after introduction of donor cells, may result
in a gradual elimination of host cells, thereby permitting human
hepatocytes to establish a "foothold" before the majority of host
hepatocyte function is eliminated.
[0061] In another non-limiting embodiment, donor hepatocytes can be
transfected with a protective gene. For example, a gene encoding an
antisense RNA or ribozyme against the cytochromes 2E1, 1A2, and/or
3A4 (CYP2E1, CYP1A2, CYP3A4, respectively), would prevent
activation of the drug acetaminophen. Metabolites of that agent
within liver cells results in hepatocyte death. Thus, donor cells
containing the transgene would have a survival advantage relative
to host cells if massive doses of acetaminophen were administered
after cell transplantation. A similar strategy would be to
transfect a mutant RNA polymerase II that is resistant to the
effects of the hepatotoxin phalloidin. Administration of phalloidin
to hosts bearing transfected human hepatocytes producing the mutant
polymerase would be protected and have a selective advantage over
host cells.
5.1.5. Confirming the Presemce of Human Hepatocytes
[0062] The presence of human hepatocytes in a host may be evaluated
by assaying for specific human markers. The presence of such
markers in a blood sample or a liver biopsy collected from the
animal (e.g., percutaneously) may be evaluated without affecting
the viability of the animal. Alternatively, the success of
chimerization may be evaluated retrospectively at necropsy.
[0063] As a specific example, the presence or absence of
immunologically distinct human albumin may be determined in a blood
or tissue sample by Western blot analysis or immunohistochemistry
using antibody specific for human, but not host, albumin (see, for
example, Wu et al., 1991, J. Biol. Chem. 266:14338-14342; Osborn
and Weber, 1982, Meth. Cell Biol. 24:97-132). An example of a
publicly available antibody specific for human albumin is Sigma
#A6684 monoclonal anti-human albumin HSA II.
5.2. Toxicology Model Systems
[0064] In particular non-limiting embodiments of the invention, a
chimeric animal prepared as set forth above may be used as a model
system for human hepatocyte function in a toxicology study to
determine the toxic effect(s) of a test agent on (i) the human
hepatocytes present in the animal and/or (ii) the host animal
itself. The chimeric animals of the invention provide the
opportunity to recapitulate, in a model system, metabolism of the
test agent by human hepatocytes, which may result in one or more
secondary compounds that may not be produced when the test agent is
exposed to non-human hepatocytes.
[0065] Because a test agent may have different effects on host
hepatocytes and human hepatocytes, it is desirable to determine the
relative proportion of human and host hepatocytes in each test
animal, for example by quantitation of the amounts of human and
non-human albumin in a serum sample. The ability of this
measurement to accurately reflect liver cell populations may be
established by correlating serum levels with hepatocyte populations
as evaluated by immunohistochemistry in liver tissue samples
obtained by biopsy or at necropsy. Once the relative proportions of
hepatocyte populations for each animal are determined, experimental
results relating to the effect of test agent may be compared with
the effect of test agent on a control non-chimeric animal which
represents a population of 100 percent host hepatocytes.
Preferably, the host hepatocytes are less sensitive to test agent
than human hepatocytes.
[0066] Accordingly, chimeric animals of the invention may be used
to evaluate the toxic effect(s) of a test agent on the viability
(survival, function) of human hepatocytes in the animal and/or the
animal as a whole by subjecting at least one and preferably a
plurality of chimeric animals and non-chimeric animals of the same
species (as controls) to incremental doses of test agent. At one or
a series of time point(s), the animal(s) may be evaluated by
standard laboratory tests to determine whether toxic effects have
occurred. Such an evaluation may include an assessment of bodily
functions, as reflected by weight and/or activity and analysis of
blood and/or urine, for example for test agent or its metabolites,
markers of liver function and/or hepatocyte viability, kidney
function, immune function, etc.. As discussed above, such
information is considered in view of the percentage of human
hepatocytes in each test animal's liver and the relative effects of
test agent on human versus host hepatocytes. Further, the
percentage of human hepatocytes may change during the course of an
experiment, for example, if the test agent is selectively toxic to
human hepatocytes so that compensatory proliferation of host
hepatocytes occurs. Accordingly, it is desirable to perform
measurements of relative quantities of one or more marker specific
for human hepatocytes at each time point; for example, the relative
amounts of human and host albumin in serum may be measured by
Western blot. At one or more time point of the study, an animal(s)
may be biopsied and analyzed for human versus host albumin gene or
gene product, or human-specific Alu repeat sequence, or sacrificed
and a complete necropsy analysis be performed, including
immunohistochemical evaluation of hepatocyte populations in the
liver.
5.3. Model Systems for Liver Diseases
[0067] In another non-limiting embodiment of the invention, an
animal having a chimeric liver may be used as a model system for
human liver disease. Such chimeric animals may be used to create
models of liver disease resulting from exposure to a toxin,
infectious disease or malignancy. The model systems of the
invention may be used to gain a better understanding of these
diseases and also to identify agents which may prevent, retard or
reverse the disease processes.
[0068] Where the chimeric animal is to be used as a model for liver
disease caused by a toxin, animals prepared as set forth above may
be allowed to mature to a point where the size of the human
hepatocyte population is substantial (e.g. has approached a
maximum), and then be exposed to a toxic agent. The amount of toxic
agent required to produce results most closely mimicking the
corresponding human condition may be determined by using a number
of chimeric animals exposed to incremental doses of toxic agent.
Examples of toxic agents include but are not limited to alcohol,
acetaminiophen, phenytoin, methyldopa, isoniazid, carbon
tetrachloride, yellow phosphorous, and phalloidin.
[0069] In embodiments where a chimeric animal is to be used as a
model for malignant liver disease, the malignancy may be produced
by exposure to a transforming agent or by the introduction of
malignant cells. The transforming agent or malignant cells may be
introduced with the initial colonizing introduction of human
hepatocytes or, preferably, after the human hepatocytes have begun
to proliferate in the host animal. In the case of a transforming
agent, it may be preferable to administer the agent at a time when
human hepatocytes are actively proliferating. Examples of
transforming agents include aflatoxin, dimethylnitrosamine, and a
choline-deficient diet containing 0.05-0.1% w/w DL-ethionine
(Farber and Sarma, 1987, in Concepts and Theories in
Carcinogenesis, Maskens et al., eds, Elsevier, Amsterdam, pp.
185-220). Such transforming agents may be administered either
systemically to the animal or locally into the liver itself.
Malignant cells may preferably be inoculated directly into the
liver.
[0070] Where the chimeric animal is to be used as a model for
infectious liver disease, the infectious agent, or an appropriate
portion thereof (e.g. a nucleic acid fragment) may be introduced
with the initial introduction of hepatocytes or after the human
hepatocytes have begun to proliferate. The infectious agent may be
administered as a free entity or incorporated into a human cell
such as a human liver cell. Examples of infectious diseases
suitable for modeling include but are not limited to hepatitis A,
hepatitis B, hepatitis C, hepatitis D, hepatitis E, malaria,
Epstein Barr infection, cytomegalovinis infection. and Yellow
Fever. For such models, it may be advantageous that the host animal
has an immune system that is intact (but for the induced tolerance
to the host cells), in that the animal's immune response to the
infectious agent and/or infected human hepatocytes may produce a
more accurate model of human liver diseases in which the immune
system plays a pathogenic role. As such, it may be desirable to
ensure that the cells/cell lysate used for tolerization not include
infectious agent or related antigens. A working example in which
the invention is used to produce a hepatitis B virus model system
is set forth below.
[0071] Further, where the infectious agent is a virus, the present
invention provides for chimeric animals comprising human
hepatocytes that contain a nucleic acid of the virus, such as the
entire viral genome or a portion thereof, or a nucleic acid encoded
by the viral genome or a portion thereof.
5.3.1. HCV Model Produced by Infectious Serum
[0072] In a particular non-limiting embodiment, the invention
provides for a chimeric animal model for hepatitis C virus
infection. Preferably, the chimeric animal is a mouse transgenic
for a gene whose product is selectively toxic to hepatocytes, such
as the albumin promoter/urokinase gene or the albumin
promoter/HSV-TK gene. Hepatitis C infection of human hepatocytes in
such mice may be produced either (i) concurrently with or
preferably (ii) after the colonizing introduction of human
hepatocytes and after the effects of the toxic transgene have
attenuated or eliminated host hepatocytes. Preferably, the chimeric
animal has, prior to infection, a liver which comprises
substantially (at least about 20 percent, preferably at least 50
percent, more preferably at least 80 percent) human
hepatocytes.
[0073] The source of infectious agent may be serum from one or more
human subject infected with HCV but not demonstrably infected with
one or more other agents that infect hepatocytes. Serum samples of
genotype Ia may be assayed for viral load by branched DNA (bDNA)
assay (Chiron, San Francisco, Calif.). Sera from non-infected
subjects and individuals with non-viral hepatitis may be used to
pseudo-infect control chimeric animals. Using standard biohazard
precautions, serum containing HCV RNA in infectious human serum, at
a titer ranging between about 10.sup.3-10.sup.7 particles per
milliliter may be injected intravenously into a chimeric transgenic
mouse about 2-4 months and preferably about 6 weeks after
colonization with human hepatocytes. Preferably, increasing amounts
of HCV RNA in infectious human serum, with the viral titer
previously determined (e.g., by National Genetics Institute, Los
Angeles, Calif. ) may be injected into a panel of such chimeric
transgenic mice. The site of injection may be the tail vein, and
the volume of serum injected may be 0.1-0.5 ml. The serum may
preferably be filter sterilized prior to administration.
[0074] Serum may be collected from the chimeric mouse (mice) and
tested to establish baseline and post-infection levels of liver
function markers such as AST (aspartate amino transferase), ALT
(alanine aminotransferase) and alkaline phosphatase. For example,
baseline and weekly post-infection levels of AST, ALT and alkaline
phosphatase in mouse sera may be determined spectrophotometrically
using kits from Sigma Chemical Co., St. Louis, Mo., where
appropriate standards are used to generate reference curves. Blood
samples may be obtained from the animals retroorbitally using
standard techniques.
[0075] The mouse (mice) may be tested for seroconversion against
HCV by testing for circulating antibody (e.g., anti-C100-3
antibody), for example using the ELISA kit available from Ortho
Diagnostics (catalog number 930740: Ortho HCV ver. 3.1 ELISA TEST
SYSTEM; Ortho Diagnostics, Raritan, N.J.). Tests for seroconversion
may be performed, for example, at weekly intervals for the first
month after infection and then monthly.
[0076] Viral load may be determined (e.g., weekly) by assay of
dilutions of serum for positive strand HCV RNA using thermostable
rTth RT-PCR performed under stringent conditions (at 70.degree. C.)
to eliminate false priming of the incorrect strand. Branched DNA
analysis may also be used, but it is not as sensitive. For positive
strand RNA analysis, the cDNA reverse primer may be: 5'-TCGCGACCCA
ACACTACTC 3' (SEQ ID NO: 2) and the forward primer may be
5'-GGGGGCGACA CTCCACCA-3' ( SEQ ID NO: 3). PCR amplification in the
absence of reverse transcriptase activity may be accomplished by
chelating manganese and magnesium ions as described in (Lanford et
al., 1995, J. Virol. 69:8079-8083). The amplified product, which
spans nucleotides 15-274 of the 5'-NTR of HCV may be quantitated by
Southern blotting using a detectably labeled probe against a region
internal to the primers.
[0077] Liver tissue obtained by biopsy or from a sacrificed animal
may be evaluated for HCV replication and for histopathological
changes. Biopsy may be performed by anesthetizing the chimeric
mouse with intramuscular injections of ketamine (40 mg/kg) and
xylazine (5 mg/kg), cleaning the abdominal area with alcohol and
betadine wipes, making a 1 cm incision in the abdominal wall to
expose the liver, and collecting a sliver (approximately 10 mg) of
liver tissue. Afterward, I OOU of sterile thrombin may be
administered locally at the biopsy site followed by application of
gel foam to inhibit bleeding, the abdominal wall may be closed with
dissolvable sutures, and the skin may be closed with nylon sutures.
Viral replication may be quantitated by measuring the amount of
negative strand template HCV RNA in liver RNA (prepared, for
example, as set forth in Chomczynski and Sacchi, 1987, Anal.
Biochem. 162:156-159), using rTth RT-PCR (Lanford et al., 1995, J.
Virol. 69:8079-8083). To assess liver histology, liver tissue may
be fixed and sectioned and stained with hematoxylin-eosin or
trichrome to evaluate, respectively, inflammation or fibrosis. A
standardized scoring method, such as Knodell scoring (Knodell et
al., 1981, Hepatology 1:531), may be used. The presence or absence
of neoplastic lesions may be evaluated.
[0078] To determine the optimum conditions for producing an HCV
infected chimeric animal, the time course of serum
aminotransferases AST and ALT, alkaline phosphatase levels, and
viral RNA loads may be plotted as a function of time and the
minimum number of viral equivalents required to sustain an
infection determined. Levels of detectable HCV RNA in the serum of
an animal may be used as an indicator of the chronicity of
infection.
[0079] Potential problems associated with the foregoing embodiment
are as follows. First, the detection of negative strand HCV
template as a measure of HCV replication may be problematic due to
the requirement for amplification techniques and the possibility of
inadvertent amplification of positive strand. The method of Lanford
et al. (supra) using stringent conditions for priming of the RT-PCR
and inactivation of the reverse transcriptase by chelation prior to
PCR of the cDNA has been shown to reduce false amplification to
{fraction (1/10)}.sup.4-{fraction (1/10)}.sup.5. Second, the fact
that mouse hepatitis virus may be found even in "pathogen free"
environments makes it desirable to confirm that host animals are
free of the virus, for example using a mouse virus screen as
available from Microbiological Associates, Inc., Rockville, Md.
(Carlson et al., 1989, J. Clin. Invest. 83:1183-1190), where
animals testing positive are not used as hosts. Third, infection
may be improved by increasing the amount of human serum used in the
inoculum.
[0080] The foregoing description may be applied to nontransgenic
mice or other animals and may be adapted, by altering volume of
inoculum and time between colonization and inoculation
proportionately, to larger animal model systems.
5.3.2. HCV Model Produced by Infectious Plasmid
[0081] In a related embodiment, infection may be introduced by HCV
plasmid (Kolykhalov et al., 1997, Science 277:570-574) complexed to
a liver-specific protein carrier, such as AsOR-PL or
AsORlysine-VSVG, where AsOR-PL is asialoorosomucoid polylysine and
AsORlysine-VSVG is asialoorosomucoid covalently linked to L-lysine
methyl ester and a synthetic 25 amino acid peptide of the VSVG
protein. The DNA-protein complex may be formed by slowly adding
protein conjugate in 25 microliter aliquots to DNA in 0.15M NaCl
with continuous vortexing at room temperature. After 30 minutes of
incubation at room temperature absorption at 260 nm, 340 nm and 400
nm may be measured to detect complex formation. Complexes may be
filter sterilized by passage through a 0.22 micron filter. About 10
micrograms of the DNA/protein complex in 0.5 milliliters sterile
saline may then be injected into the tail vein of a mouse.
5.3.3. HCV Model Produced by Transplanting Infected Hepatocytes
[0082] As an alternative to producing HCV infection by inoculation
with infected serum, infection may be produced by transplanting HCV
infected hepatocytes into a chimeric animal. Although the infected
hepatocytes may be introduced during colonization with human cells,
it is preferred that they be introduced into chimeric livers having
a substantial population of human hepatocytes. Preferably, the
chimeric animal is a mouse transgenic for a gene whose product is
selectively toxic to hepatocytes, such as the albumin
promoter/urokinase gene or the albumin promoter/HSV-TK gene.
[0083] Infected human hepatocytes may be obtained as described in
Lieber et al., 1996, J. Virol. 70:8782-8791. Using appropriate
pathogen-containment procedures, human liver specimens may be
obtained from HCV-infected liver transplant recipients. An apical
piece of liver covered on three sides by capsule may be perfused
with buffer without calcium and then with collagenase in perfusion
buffer with calcium. Hepatocytes may then be pelleted by low speed
centrifugation. Non-parenchymal cells may be separated from
parenchymal hepatocytes by metrizamide gradient centrifugation. The
viability of isolated hepatocytes may be evaluated by trypan blue
exclusion. Hepatocytes may be resuspended in Williams medium at
about 10.sup.7 cells per milliliter.
[0084] The infected hepatocytes may then be introduced into the
liver of a chimeric animal such as a chimeric trausgenic mouse
having a liver which comprises substantially (at least about 20
percent, preferably at least 50 percent, more preferably at least
80 percent) human hepatocytes. The infected hepatocytes may be
introduced by intrasplenic injection. Where the animal is a mouse,
hepatocytes may be achieved by anesthetizing the animal with
ketamine (90 mg/kg)/xylazine (10 mg/kg), and then, under aseptic
conditions, making a 2-3 millimeter incision in the left paracostal
area, exposing the spleen. The spleen may then be exteriorized and
infected hepatocytes may be injected slowly into the spleen
parenchyma. Gel foam may be used to achieve hemostasis, the spleen
may be restored into the body cavity, and the wound may be sutured
closed. Monitoring of the resulting infected animals for
serconversion, viral load, serum levels of protein markers of liver
function, and histopathology may be performed as described in
section 5.3.1.. Further, these methods may be applied to
nontransgenic mice and may be adapted for use in larger
animals.
5.3.4. Use of HCV Models
[0085] Chimeric animal models of HCV infection may be used not only
to study the biology of HCV, but also to identify agents that may
prevent or inhibit HCV infection and/or replication. For example,
to determine whether a test agent inhibits infection by HCV, the
effect of the agent on preventing infection when administered prior
to or contemporaneously with injection of infected serum may be
evaluated. Similarly, the effect of a test agent administered
during the course of infection may be assessed. Parameters useful
in determining the effectiveness of test agent would include
whether and when the test animal seroconverts with respect to HCV,
the viral load, the ability of serum from the animal to infect
other animals, blood levels of proteins/enzymes associated with
liver function and/or hepatocyte viability, and liver
histology.
5.4. Chimeric Animals as a Source of Hepatocytes for Liver
Reconstitution
[0086] The present invention further provides for the use of
chimeric animals as a source of human hepatocytes for liver
reconstitution in a second host subject. Such reconstitution may be
used, for example, to (i) produce a "next generation" chimeric
non-human animal; (ii) introduce genetically modified hepatocytes
for "gene therapy" of the second host subject; or (iii) replace
hepatocytes lost as a result of disease, physical or chemical
injury, or malignancy in the second host. Human hepatocytes
collected from a chimeric animal are said to be "passaged".
[0087] For any of these applications, liver tissue from a chimeric
animal may be used to produce a cell suspension and then human
hepatocytes may be separated from non-human hepatocytes and other
cells. The liver tissue may be processed as set forth above to
produce a suspension of hepatocytes. As a non-limiting specific
example, where the chimeric animal is a mouse or rat, hepatocytes
may be prepared by the following method, adapted from Seglen, 1976,
"Preparation of rat liver cells", Methods Cell Biol. 13:29.
Briefly, a chimeric mouse or rat may be anesthetized with
ketamine/xylazine, its abdomen may be shaved and decontaminated,
the peritoneal cavity may be opened by incision, the inferior vena
cava may be cannulated, the portal vein may be divided and the
suprahepatic vena cava may be ligated. Then, the liver may be
perfused in situ with calcium free balanced salt solution at 5
ml/min for five minutes at 37.degree. C., followed by perfusion
with 0.05% collagenase (e.g., type IV, from Sigma Chemical Co.) in
1% albumin and balanced salt solution for 20 minutes. The liver may
then be transferred to a Petri dish, and minced to produce a cell
suspension, from which hepatocytes may be collected by passage
through a 60-80 micron nylon mesh. The collected cells may then be
washed three times in RPMI 1640 or Williams E medium with 10% fetal
bovine serum, and then centrifuged at 35.times.g for five minutes
at 4.degree. C. Hepatocytes may be purified through a metrizamide
gradient and resuspended in RPMI 1640 or Williams E medium.
[0088] Human hepatocytes may be separated from non-human cells
using fluorescence activated cell sorting techniques and an
antibody which selectively binds to human hepatocytes, for example
but not by way of limitation, an antibody that specifically binds
to a class I major histocompatibility antigen. Suitable antibodies
would include but not be limited to anti-human HLA-A,B,C,
Pharmingen catalogue # 32294X or #32295X, FITC mouse .kappa.b,
PharMingen catologue #06104D (PharMingen, San Diego, Calif.) See,
for example, the procedure described in Markus et al., 1997, Cell
Transplantation 6:455-462.
[0089] Human hepatocytes may be passaged through cell
transplantation of tolerized host animals, using the techniques set
forth above. In this manner, cells obtained from an initial human
donor may be utilized in a multitude of chimeric animals and over
an extended period of time, potentially reducing the variability
that may be encountered in chimeric animals produced using
hepatocytes obtained from diverse hosts.
[0090] Passaged human hepatocytes may also be used for gene therapy
applications. In the broadest sense, such hepatocytes are
transplanted into a human host to correct a genetic defect. The
passaged hepatocytes need not, but are preferably derived
originally from the same individual who is to be the recipient of
the transplant. However, according to the invention, hepatocytes
from a different individual may alternatively be used.
[0091] As a specific, non-limiting example, a patient suffering
from intermittent acute porphyria, caused by a genetic defect in
the expression of uroporphyrinogen I synthase, may benefit from
transplantation of human hepatocytes harvested from a chimeric
animal of the invention, where the transplanted cells are
genetically normal in their expression of that enzyme. The
recipient would be "matched" for transplantation antigens with the
original donor, or be treated with immunosuppressive therapy. For
such applications, chimeric animals prepared from a wide diversity
of individual donors could provide the advantage of constituting a
"living library" of differentiated bepatocytes having various
transplantation antigen profiles, thereby obviating the need for
waiting until liver tissue from a genetically suitable donor
becomes available.
[0092] Preferably, however, the original donor and eventual
recipient of passaged hepatocytes are the same person, thereby
eliminating the need for immunosuppression. For gene therapy
applications, (i) hepatocytes may be harvested from the subject,
(ii) the desired genetic construct may be introduced into those
hepatocytes, (iii) the resulting genetically engineered human
hepatocytes may be used to tolerize a host animal to their
presence, (iv) construct-carrying hepatocytes may be introduced
into the tolerized animal such that its liver is colonized, and
then, once expanded in number, (v) the transgenic hepatocytes may
be harvested from the chimeric animal and (vi) reintroduced into
the subject. A genetic construct may be introduced into the human
hepatocytes by any standard method, including, but not limited to,
transfection with naked DNA, microparticles or liposomes, or
infection with a viral vector, such as an adenoviral vector, an
adeno-associated vector, or a retroviral vector. Hepatocytes used
for colonization may be enriched for cells containing the desired
construct, for example, by selection by culture conditions,
antibody/FACS methods, etc. which eliminate cells lacking the
construct.
[0093] Alternatively, the hepatocytes may be used to colonize the
liver of a tolerized animal prior to or contemporaneous with the
introduction of the desired transgene via a gene therapy vector.
This approach may be more problematic because the host animal could
develop an immune response directed toward either the vector or
vector-transformed hepatocytes.
[0094] In further embodiments, human hepatocytes passaged through a
chimeric animal of the invention may be used to reconstitute liver
tissue in a subject as a prelude or an alternative to liver
transplant. As a specific non-limiting example, a subject suffering
from progressive degeneration of the liver, for example, as a
result of alcoholism, may serve as a donor of hepatocytes which are
then maintained, through one or several generations, in one or more
chimeric animal. As a result of maintenance in such animal(s), the
number of hepatocytes is expanded relative to the number originally
harvested from the subject (it may be preferable to use larger
animals to produce greater numbers of cells). At some later date,
when the subject's liver has deteriorated to a medically hazardous
condition, hepatocytes passaged in the chimeric animal(s) may be
used to reconstitute the subject's liver function. As a second
non-limiting example, passaging hepatocytes may be used not only to
expand the number of hepatocytes but also to selectively remove
hepatocytes that are afflicted with infectious or malignant
disease. Specifically, a subject may be suffering from hepatitis,
where some but not all of the hepatocytes are infected and infected
hepatocytes can be identified by the presence of viral antigens on
the cell surface. In such an instance, hepatocytes may be collected
from the subject, and non-infected cells may be selected for
passaging in one or more chimeric animal, for example by FACS.
Meanwhile, aggressive steps could be taken to eliminate infection
in the patient. Afterward, the subjects liver tissue may be
reconstituted by hepatocytes passaged in a chimeric animal. An
analogous method could be used to selectively passage non-malignant
cells from a patient suffering from a primary or secondary (e.g.
metastatic) liver malignancy. Thus, the chimeric animals of the
invention may be used as a means of purging unwanted hepatocytes
from a human subject.
6. EXAMPLE
Prepartaion of Rats Having Chimeric Livers
6.1. Survival of Human Hepatocytes in Rats tolerized by
Intraperitoneal Injection
[0095] To tolerize hosts for hepatocyte transplantation, human
hepatocytes obtained from Clonetics Corp. were suspended at a
concentration of 10.sup.6 cells/ml saline and sonicated.
Laparotomies were performed to expose the gravid uteri of pregnant
rats and sterile filtered sonicates equivalent to 10.sup.4
hepatocytes in 10 .mu.l were injected into groups of three fetuses
each. A control received only the same volume of normal saline.
Within 24 hrs of birth, transplantation of hepatocytes were
performed by the method of Marucci et al., 1997, Hepatol. 26:
1195-1202. An incision was made at the left paracostal area, the
spleen was visualized and 2.times.10.sup.7 hepatocytes in 100 .mu.l
sterile medium were injected slowly into the spleen. To evaluate
the status of immune tolerance, two experiments were performed: a)
mixed lymphocyte assays and b) repeat transplantation
challenges.
6.1.1. Mixed Lymphocyte Assays
[0096] For mixed lymphocyte assays, spleens from intrafetally
injected animals were removed six weeks after birth, and spleen
cells were isolated as described by Henry and Watson (1980,
"Preparation of Spleen Cells" section 2.9, in Selected Methods in
Cellular Immunology, Mishell and Shiigi, eds., W H Freeman and Co.,
p.65). Assays were performed according to the method of Bradley
(1980, "Mixed Lymphocyte Responses" section 6.3, in Selected
Methods in Cellular Immunology, Mishell and Shiigi, eds., W H
Freeman and Co., p. 162) in which spleen cells were used as
responder cells and human hepatocytes (identical to those
transplanted but treated with 2000R of X-irradiation to prevent
proliferation) were used as stimulator cells. Human hepatocytes
should not stimulate lymphocyte proliferation in spleen cells from
immunotolerant animals, but should in cells from non-tolerant
animals.
[0097] Spleen cells, 7.times.10.sup.5 cells per assay, from fetuses
previously injected intraperitoneally as described above with
hepatocyte lysates alone, lysates followed by hepatocytes after
birth, or controls were mixed with 3.times.10.sup.5 X-irradiated
simulator hepatocytes. Controls consisted of spleen cells from
saline treated fetuses alone, and those same cells plus irradiated
spleen cells from non-treated animals. Cells were pulsed with 1
.mu.C.sup.3[H]-thymidine (specific activity 5 Ci/mmole) at
37.degree. C. for 72 hours, and then harvested by TCA precipitation
onto Whatman glass fiber filters, washed and scintillation counted.
All assays were performed in quadruplicate, and the entire
experiment done in duplicate. The results are expressed as means
.+-.S.D. in units of cpm/10.sup.6 responder cells in FIG. 1.
[0098] FIG. 1 shows that spleen cells from animals that did not
receive hepatocyte lysate when they were fetuses incorporated
approximately 5,200 cpm/10.sup.6 cells when stimulated with
irradiated human hepatocytes. In contrast, stimulation of spleen
cells derived from animals that did receive human hepatocyte lysate
when they were fetuses was significantly less (less than 1000
cpm/10.sup.6 cells [p<0.002]) than that observed using cells
from animals that did not receive lysate. In fact, cells derived
from animals injected with lysates resulted in stimulation that was
no more than background levels of spleen cells alone. Irradiated
hepatocytes alone (without spleen responder cells) had no
significant radioactive incorporation confirmning that the
irradiation substantially blocked any contribution to the observed
radioactive uptake.
[0099] These data indicate that spleen cells removed 6 weeks after
birth from rats previously injected as fetuses with human
hepatocyte lysates are not significantly stimulated to proliferate
by the presence of human hepatocytes. At this time point, human
albumin is still strongly detectable in serum in the tolerized
animals. Together, the data indicate that immune tolerance to human
hepatocytes was achieved.
6.1.2. Rechallenge with Additional Hepatocyte Transplantations
[0100] As further evidence for immune tolerization, groups of rats
previously tolerized and transplanted as described above were
subjected to a repeat transplantation. If the rats were not
rendered immunologically tolerant to human hepatocytes, a second
transplantation of human cells would be expected to evoke an
anamnestic response and rapid rejection of those cells. To evaluate
that possibility, rats tolerized intrafetally and transplanted with
human hepatocytes at birth as described above were given a repeat
transplantation of 2.times.10.sup.6 cells 6 weeks after the first.
Serum was assayed for human serum albumin using a specific
anti-human albumin antibody and analyzed by Western blots as shown
in FIG. 2.
[0101] In FIG. 2, lane 1 contains 40 ng of human serum albumin
standard, and lane 2 shows that there was human albumin still
present in substantial concentration in serum at 6 weeks after an
initial cell transplantation. After a second injection of human
cells the amount of serum albumin increased 24 hrs later (lane 3),
and continued to rise at least 8 days after the repeat dose of
cells (lane 4). Animals that had not been tolerized (received only
a fetal injection of saline) had no detectable human serum albumin
even after a repeat transplant of human hepatocytes under identical
conditions and assayed at the same time point (8 days; lane 5).
These data suggest that human hepatocytes did not survive in
non-tolerized animals. In contrast, in those animals that were
tolerized, human hepatocytes not only survived each of two
successive inoculations, but also maintained function as evidenced
by serum albumin production.
6.1.3. Survival of Human Hepatocytes
[0102] Seventeen day old normal Sprague-Dawley rat fetuses were
given lysates of 0.5.times.10.sup.5 human hepatocytes by
intrauterine inoculation into the peritoneum under
trans-illumination. Following birth, 2.times.10.sup.5 normal human
hepatocytes were injected into the spleen. This is known to result
in near total migration of transplanted hepatocytes to the liver
(Attavar, et al., 1997, Hepatol. 26: 1287-1295). At weekly
intervals, animals were bled via their tail veins and human serum
albumin was detected as a function of time by Western blot analysis
using a specific affinity purified rabbit anti-human albumin
antibody( FIG. 3). At the conclusion of the study, animals were
sacrificed and liver slices stained with anti-human albumin
antibody and developed with a Texas red secondary antibody. Cells
were visualized using a Zeiss confocal scanning microscope, model
CLSM4 10 and images were captured as shown in FIG. 4.
[0103] FIG. 3 shows a representative Western blot of the collected
rat sera, where lane 1 contains 10 ng standard human serum albumin,
lane 2 contains 10 ng standard rat albumin, lane 3 contains rat
serum 1 week after intrasplenic injection into the tolerized rat (1
day after birth), lane 4 contains serum from the same rat as lane 3
but after 2 weeks, lane 5 same as lane 3, but after 3 weeks. Lane 1
shows that the anti-human albumin antibody reacted with standard
human serum albumin, but not with rat albumin, (lane 2) (nor mouse
albumin, data not shown). Lanes 3, 4, and 5 showed bands
corresponding in mobility to albumin detectable at about 10 ng/50
.mu.g total serum protein. This level remained stable or increased
through at least week 3. Transplantation of human IMR-90
fibroblasts under identical conditions failed to produce any
detectable human serum albumin.
[0104] FIGS. 4A-D shows a representative immunofluorescence study
of a liver section taken from 1 of 4 rats 3 weeks after injection
with human hepatocyte lysate in utero, followed by intrasplenic
injection of human hepatocytes. Immunocytochemistry was performed
with primary antibody for human albumin, or rat albumin as a
control, and Texas red-coupled secondary antibody. Panel A shows
anti-human albumin antibody staining of rat liver without human
hepatocyte transplantation. Panel B shows rat livers 3 weeks
following injection with human hepatocyte lysate in utero and
intrasplenic injection of human hepatocytes (1 day after birth)
stained with anti-human albumin antibody and Texas Red second
antibody. Panel C shows the same section as depicted in B, but
without second antibody. Panel D shows a section of control rat
liver after only intrauterine injection of human hepatocyte lysate
developed with anti-human albumin antibody and Texas Red second
antibody. Anti-human albumin staining of liver transplanted with
human hepatocytes, but without prior injection with hepatocyte
lysate was essentially the same as that shown in Panel A. There was
no anti-human albumin staining of normal (non-transplanted rat
liver) in Panel A. Cells with fluorescent cytoplasm are seen in
Panel B, after both in utero lysate injection and human hepatocyte
transplantation. This staining was not due to intrinsic
fluorescence as there was no signal without second antibody as
shown in Panel C. Finally, Panel D shows that the fluorescence
could not be due to human albumin from the hepatocyte lysate alone.
All 3 other animals injected with human hepatocyte lysate in utero
and intrasplenic injection of human hepatocytes showed similar
results.
[0105] FIG. 5 is a photomicrograph of the same section of rat liver
as depicted in FIG. 4B, 3 weeks after intrasplenic injection of
human hepatocytes, here stained with hematoxylin and cosin. The
human hepatocytes cannot be distinguished from the rat cells, and
there appears to be no inflammation or other evidence of
rejection.
[0106] FIGS. 6A-D shows the results of an immunofluorescence study
performed six weeks after cell transplantation in tolerized
animals. Aggregates of cells staining positive for human albumin
were present in a tolerized rat that had received a human
hepatocyte transplant, panel A. The same section without second
antibody, panel B; or no antibody at all failed to produce a
fluorescent signal, panel C. Furthermore, a liver section from a
non-tolerized animal that had received a human hepatocyte
transplant also produced no stained cells after 6 weeks, panel D.
Scanning many fields also showed that most of the positive cells at
6 weeks were in groups, while sections taken at 2 weeks showed
scattered single cells, predominantly. Because the injected cell
suspensions were predominantly single cells, and because the
fluorescence data at 2 weeks showed predominantly isolated single
cells, the finding of groups of cells at 6 weeks suggests that the
human cells transplanted into tolerized rats not only survived, but
proliferated to some extent in the host liver environment.
6.1.4. Induction of Tolerance to Human Hepatocytes by Intrathymic
Injection in Neonatal Rats
[0107] Injection of human hepatocytes was performed according to
the method of Fabrega et al., 1995, Transplantation 59:1362-1364.
10.sup.2 human hepatocytes in 5 .mu.l sterile medium were injected
into the thymuses of 1-2 hour old newborn rats. Five days following
intrathymic injection, 10.sup.5 human hepatocytes in 200 .mu.l
sterile medium was injected into the spleen (Marucci et al., 1997,
Hepatol. 26:1195-1202). Blood was collected by tail vein puncture
at the time of intrathymic injection and at weekly intervals
following intrasplenic injection of hepatocytes and assayed for
human albumin by Western blot analysis.
[0108] A representative Western blot of serum from one of 6 animals
after intrathymic tolerization, followed by transplantation of
human hepatocytes, is shown in FIG. 7. Lane 1 contains 10 ng
standard human albumin; lane 2 contains 10 ng standard rat albumin
and lanes 3-7 contain sera taken at the indicated times after
transplantation of human hepatocytes. The data show that human
albumin production increased until about 5 weeks and then remained
stable. The other five animals had shown similar results. 7.
EXAMPLE
A Model for Hepatitis B Virus Infection
7.1 Purification of HBV Particles form HEPG2 2.2.15 Cells
[0109] To test the possibility of infection of human hepatocytes in
vivo, infectious HBV particles were prepared from the HepG2 2.2.15
cell line (obtained from Dr. George Acs, Mt. Sinai School of
Medicine, N.Y.) which contains an integrated tandem repeat genome
of HBV ayw strain. The cell line actively secretes infectious virus
into the medium (Sells et al., 1988, J. Virol 62:2836-2844).
Culture medium from HepG2 2.2.15 was clarified by centrifugation at
5,000 rpm, 4.degree. C. for 30 min. The supernatant was layered on
a 5 ml 25% sucrose cushion in TEN buffer (150 mM NaCl, 20 mM
Tris-HCl, pH 7.4) and centrifuged at 25,000 rpm for 16 hrs at
4.degree. C. The resulting pellet was resuspended in TEN buffer,
applied onto a 20-50% continuous CsC1 gradient, and centrifuged at
35,000 rpm for 16 hrs at 4.degree. C. Fractions with buoyant
densities between 1.24 g/ml and 1.28 g/ml containing HBV virus were
collected, dialyzed against TEN and sterile filtered through 0.22
.mu. filters.
7.2 Infection of Human Hepatocytes Transplanted into Tolerized Rat
Hosts
[0110] To determine whether human hepatocytes in rat liver could be
infected with human hepatitis virus, one week after human
hepatocyte transplantations, tolerized rats were given intrasplenic
injections of 10.sup.4 hepatitis B viral particles (purified from
HepG2 2.2.15 cells as described above) in 50 .mu.l. Control animals
tolerized but without human hepatocyte transplants, and tolerized
transplanted animals without HBV were used as controls. Liver
sections were removed by liver biopsy at 1 week, and partial
hepatectomy at 6 weeks and 14 weeks post HBV treatment and analyzed
as described below.
7.3 Identification of the Presence of Human Liver Cells and HBV
Infection
[0111] To identify and quantify human hepatocytes in host liver,
immunohistochemical staining for human albumin was used.
Visualization of cells infected with HBV was similarly achieved by
immunohistochemical staining for Hepatitis B Surface Antigen
(HBsAg). Liver tissues were flash frozen in liquid nitrogen
immediately following removal. Samples were prepared according to
the method of Osborne and Weber (1982, Meth. Cell Biol. 24:97-132)
with minor modifications. Liver cryosections 6 .mu.m thick were
fixed in 4% paraformaldehyde for 15 min at 25.degree. C., and
washed 3 times with phosphate buffered saline (PBS) pH 7.2. Liver
sections were quenched with 10% non-fat milk in PBS for 30 min at
25.degree. C. followed by successive incubations with {fraction
(1/1000)} dilution of rabbit anti-human albumin (Sigma). Identical
sections were stained with {fraction (1/1000)} dilution of goat
anti-HBV surface antigen (DAKKO). Each section was incubated for 2
hrs at room temperature. Between each primary antibody incubation,
liver sections were exposed to 10% non-fat milk in PBS containing
0.05% NP-40 and 3 times with 10% non-fat milk in PBS alone. Texas
red-conjugated anti-rabbit antibody ({fraction (1/100)} dilution)
was used to develop anti-human albumin and FITC conjugated
anti-goat ({fraction (1/100)} dilution) antibody was used to
develop anti-HBsAg. Sections were incubated with the second
antibodies for 30 min at 25.degree. C. Following 3 washes with 10%
non-fat milk in PBS, and a final PBS wash, sections were treated
with anti-fade 2.5% 1/4-diazobicyclo-[2.2.2]-octane (DABCO)
(Sigma), covered with cover slips and stored at 20.degree. C. in
light-proof boxes. Immunofluorescence of liver sections were
visualized with Zeiss Scanning laser confocal microscope (Model
LSM-410, Carl Zeiss, New York) using 63.times. objective. Frozen
sections of chimeric liver were incubated with antibodies against
human albumin and HBV surface antigen to detect the presence of
human albumin and HBV proteins. Human albumin was detected with
Texas red conjugated secondary antibody and HBV surface antigen
detected with the use of FITC-conjugated second antibody. FIGS.
8A-F clearly demonstrates that both human albumin, left panels 8A,
8C and 8E, and HBV surface antigen, right FIGS. 8B, 8D, and 8F,
were clearly detectable within cells in livers of tolerized rats
transplanted with human hepatocytes followed by HBV treatment at 1
week (FIGS. 8A and 8B); 6 weeks (FIGS. 8C and 8D); and 14 weeks
(FIGS. 8E and 8F). Furthermore, the staining for HBsAg appears to
present only in cells that also stained for albumin. However, many
cells that contained albumin were also positive for HBV. In a liver
sample obtained 14 weeks after injection with HBV, FIGS. 9A-H shows
that albumin stained cells were only found in animals that had been
tolerized and had received transplanted human hepatocytes (FIGS. 9A
and 9C). Animals without human cell transplants that received HBV
had no albumin signal (FIG. 9E). Only tolerized animals that
received human hepatocyte cell transplants prior to infection had
HBsAg staining (FIG. 9B). As expected, the same animals without HBV
infection failed to show any HBsAg staining indicating that the
signal seen in FIG. 9B was not a non-specific artifact.
Furthermore, animals that had no human cells, but did receive HBV
injection also had no HBsAg signal (FIGS. 9E and 9F). The data
indicate that injected virus had already been completely cleared by
the time the liver sample was obtained and that the signal observed
in row 1 was due to the infection of cells, and was not an artifact
of circulating injected HBV. FIGS. 9G and 9H show that, in the same
samples as depicted in FIGS. 9A and 9B, but without primary
antibody, there was no signal corresponding to either albumin (FIG.
9G) or HBsAg (FIG. 9H), indicating the staining was not due to
non-specific binding of second antibody to the tissue
specimens.
7.4 Histological Evaluation of Livers Exposed to HBV
[0112] To determine whether exposure of human hepatocyte
transplants to HBV could result in a histological hepatitis in
vivo, serial slide sections were stained with hematoxylin and eosin
and examined in a blinded fashion by a pathologist. The results,
discussed below, are shown in FIGS. 13A-C and 14A-C.
7.5 Assessment of Function of Transplanted Human Hepatocytes
[0113] Because albumin synthesis is a selective function of
hepatocytes, levels of albumin mRNA were used to determine the
activity of transplanted human cells in host liver. To accomplish
this, specific primers for human and rat (control) albumin were
used. HBV mRNA was detected similarly. Total RNA was extracted from
100 mg liver tissue with acid guanidinium thiocyanate according to
the method of Chomczynski and Sacci (1987, Anal. Biochem. 162:
156-159). Poly A+RNA was isolated from total RNA by the method of
Aviv and Leder (1972, Proc. Natl. Acad. Sci. USA 69: 1408-1412).
RNA was reverse transcribed and amplified by polymerase chain
reaction according to the method of Berchtold (1989, Nucl. Acids
Res. 17: 453) with some modifications. Briefly, 10 .mu.g total RNA
or 1 .mu.g polyA+RNA was mixed with 2 pmol of random primer
(Gibco/BRL, Gaitherburg, Md.) at 70.degree. C. for 15 min, and then
cooled on ice. Two hundred units of Moloney Murine Leukemia Virus
reverse transcriptase (Gibco/BRL, Gaithersburg, Md.) was used to
reverse transcribe the RNA for 50 min at 42.degree. C. Reaction was
stopped by heating to 70.degree. C. for 15 min, after which the
cocktail was chilled on ice and treated with 10 .mu.g RNase A at
37.degree. C. for 20min.
[0114] From the total cDNA, polymerase chain amplification of human
albumin was performed using, as antisense primer,
5'-CCTTGGTGTTGATTGCCTTT- GCTC-3' (SEQ ID NO: 4) and as sense
primer, 5'-CATCACATCAACCTCTGTCTGACC-3' (SEQ ID NO: 5). If present,
the albumin cDNA would generate a characteristic 315 bp fragment of
the human albumin gene spanning nucleotides 176-491. For rat
albumin, an antisense primer 5'-ATAGTGTCCCAGAAAGCTGGTAGGG-3' (SEQ
ID No: 6) and a sense primer: 5'-CGGTTTAAGGACTTAGGAGAACAGC-3' (SEQ
ID No: 7) were used to generate an expected 400 bp fragment of the
rat albumin gene spanning nucleotides 104-504. To search for the
presence of HBV in liver, an antisense primer
5'-ATCTTCTGCGACGCGGCGATGGAGATC-3' (SEQ ID No: 8) and a sense primer
5'-CTCTGCTGGGGGGAATTGATGACTCTAGC-3' (SEQ ID NO: 9) were used to
generate a characteristic 355 bp fragment of the ayw HBV genome
spanning nucleotides 2079-2434. One third of the total cDNA was
mixed with 100 pmol of amplification primers and 2.5 U Taq
polymerase and amplified at 1 cycle at 94.degree. C. for 3 min,
then for 38 cycles of 94.degree. C. for 1 min, 55.degree. C. for 1
min, and 72.degree. C. for 1 min; and then 1 cycle at 72.degree.
for 5 min. The PCR products were analyzed on 1.0% agarose gels in
Tris-Borate-Acetate buffer.
[0115] FIG. 10, lanes 1 and 2 show that the RT-PCR products of
albumin from rat and human can be completely distinguished from
each other based on electrophoretic mobility. Lane 4 shows RNA from
HepG2 2.2.15 cells demonstrating a strong level of albumin
synthesis in these cells. In tolerized animals that had human
hepatocyte transplants, the rat albumin signal had the same
intensity in cells infected with HBV (lanes 5 and 6) compared to
those which were not (lanes 7 and 8). However, the human albumin
mRNA signal in cells infected with HBV (lanes 5 and 6) appeared to
be increased compared to non-infected cells (lanes 7 and 8). As
expected, control animals that had no human hepatocyte transplants,
but were administered HBV, had no detectable human albumin signal
(lanes 9 and 10).
[0116] A time course of the levels of human albumin and HBV message
is shown in FIG. 11. Compared to albumin RNA from hunan liver cells
in the upper panel, lane 3, and from HepG2 2.2.15 cells lane 4,
human albumin messenger RNA signal at 387 bp was easily detectable
at one week after HBV infection, lane 5; with at least equal signal
from week 6 and 14, lanes 6 and 7, respectively.
[0117] FIG. 11, bottom panel shows that HBV RNA could be detected
in livers by the presence of a 355 bp band at 1 week after
injection, lane 5. The intensity appeared to increase slightly at 6
weeks, lane 6; and remained strong at 14 weeks, lane 7, after HBV
inoculation. The same primers were used to amplify HBV RNA from a
human liver cell line HepG2 2.2.15 that continually produces HBV
and was used as the source of HBV viral particles for the
infections. FIG. 12 shows that HBV RNA could not be detected in
livers of tolerized rats that did not receive hepatocyte
transplantation, but received HBV (lanes 9, 10), indicating that
the signal was not due to residual injected HBV.
[0118] FIGS. 13A-C depicts slides of livers from tolerized rats,
transplanted with human hepatocytes and infected with HBV, at low
power (20.times.). FIG. 13A shows that liver, one week after
infection, has normal architecture for that stage in life and no
evidence of inflammatory cell infiltration. However, after 6 weeks
(FIG. 13B), foci of necrosis and mononuclear cell infiltrates can
be seen. FIG. 13C shows substantial mononuclear inflammation after
14 weeks with an increase in Kupffer cells as well. FIGS. 14A-C
shows that at high power (40.times.), the infiltrates are more
easily seen to be to be mononuclear cells surrounding areas of
necrosis at 6 weeks (FIG. 14B). At 14 weeks, the inflammation
extends into the surrounding parenchyma (FIG. 14C).
7.6. Detection and Quantitation of HBsAg in Rat Serum
[0119] To follow the course of infection, levels of HBsAg in rat
serum were measured using an EIA kit for HBV surface antigen
(Abbott Labs, Abbott Park, Ill.) according to the manufacturer's
protocol. Briefly, 10 .mu.l serum in 190 I1 saline was mixed with
anti-HBs (mouse) monoclonal antibody coated beads and 50 .mu.l of
horseradish peroxidase conjugated anti-mouse secondary antibody and
incubated at room temperature for 16 hours. Then the incubation
solution was removed and the beads were washed six times with 10 ml
distilled water, and the beads were transferred to clean assay
tubes and incubated with 300 .mu.l of freshly prepared
o-phenylenediamine substrate and quantitated using a
spectrophotometer at 492 nm. Assays were done in triplicate and the
results (see Tables 1, 2 and 3, third column) were expressed as
means .+-.S.D. in units of pg/ml serum.
7.8. Detection of Serum Alanine Aminotransferase (ALT)
[0120] To determine whether HBV infection was associated with any
liver damage, serum was collected from rats as a function of time
after injection, and serum ALT values determined in triplicate from
10 .mu.l serum using a commercial ALT detection kit (Sigma). All
assays were done in triplicate and results are expressed as means
.+-.S.D. International Units (IU)/ml.
[0121] Group 1 animals were treated by (i) intrafetal injections
with human hepatocyte lysates into the peritoneums at 17 days
post-conception; (ii) intrasplenic saline injection at birth; and
(iii) one week later, purified HBV harvested from a human hepatoma
cell line was administered by intrasplenic injection.
1TABLE 1 Time Post-HBV Injection (days) ALT (IU/L) HBsAg (pg/ml) 0
28 .+-. 15 Not detectable 1 44 .+-. 20 Not detectable 8 22 .+-. 12
Not detectable 10 25 .+-. 10 Not detectable
[0122] As shown in Table 1, animals that received no tolerization
or human hepatocytes, but where injected with HBV had no
significant changes in ALT as a measure of liver cell damage, or
detectable HBsAg in the serum even as soon 1 day after injection of
HBV through at least 10 days. The data confirm that HBV does not
cause hepatic damage in rats and that the virus is rapidly cleared
from the circulation.
[0123] Animals in Group 2 were treated by (i) intrafetal injection
with human hepatocyte lysate into the peritoneums at 17 days
post-conception; (ii) intrasplenic injection of 2 million human
primary hepatocytes at birth; and (iii) 1 week post hepatocyte
transplantation, saline was injected intraplenically.
2TABLE 2 Time Post-HBV Injection (days) ALT (IU/L) HBsAg (pg/ml) 0
25 .+-. 15 Not detectable 1 22 .+-. 20 Not detectable 8 30 .+-. 5
Not detectable 10 25 .+-. 16 Not detectable
[0124] As shown in Table 2, animals tolerized with human
hepatocytes that received human hepatocyte tansplants, but no HBV,
also had normal ALT and undetectable HBsAg throughout the 10 days.
Thus, without inoculation with HBV, there was no serological
evidence of hepatotoxicity or circulating HBV.
[0125] Animals in Group 3 were treated by (i) intrauterine
injection into the peritoneum at 17 days post-conception with
primary human hepatocyte lysate; (ii) intrasplenic injection of 2
million human primary hepatocytes at birth; and (iii) HBV was
injected intrasplenically at 1 week post-hepatocyte
transplantation.
3TABLE 3 Time Post-HBV Injection (days) ALT (IU/L) HBsAg (pg/ml) 0
22 .+-. 15 Not detectable 1 47 .+-. 10 0.2 .+-. .01 4 86 .+-. 15
0.1 .+-. .05 8 132 .+-. 15 0.4 .+-. 0.1 10 179 .+-. 20 0.5 .+-.
.15
[0126] As shown in Table 3, in this group which was tolerized and
received human hepatocytes and HBV, ALT levels were normal until
day 4 when the level doubled to 86 IU/L. By day 10, the ALT had
doubled again to 179 IU/L. The HBsAg was detectable at 0.1-0.2
pg/ml through day 4. However, the levels doubled to 0.4 and reached
0.5 pg/ml day 10. These data suggest that viral antigen and likely
viral levels increase early in the process, and are followed by
liver cell damage. This is supportive of an inflammatory process
triggered by the injection of HBV, but only in animals that have
human hepatocytes.
[0127] Various publications are cited herein, the contents of which
are hereby incorporated by reference in their entireties.
Sequence CWU 1
1
11 1 30 DNA Mus musculus 1 tgtgcttatg tagccatcca gcgagtcccc 30 2 19
DNA Hepatitus C virus 2 tcgcgaccca acactactc 19 3 18 DNA Hepatitus
C virus 3 gggggcgaca ctccacca 18 4 24 DNA Homo sapiens 4 ccttggtgtt
gattgccttt gctc 24 5 24 DNA Homo sapiens 5 catcacatca acctctgtct
gacc 24 6 25 DNA Rattus ratus 6 atagtgtccc agaaagctgg taggg 25 7 25
DNA Rattus ratus 7 cggtttaagg acttaggaga acagc 25 8 27 DNA Rattus
ratus 8 atcttctgcg acgcggcgat ggagatc 27 9 29 DNA Hepatitus B virus
9 ctctgctggg gggaattgat gactctagc 29 10 25 DNA Hepatitus B virus 10
gccggtctgg agcaaagctc atcgg 25 11 24 DNA Hepatitus B virus 11
ggcggtgtct aggagatctc tgac 24
* * * * *