Note: Descriptions are shown in the official language in which they were submitted.
W094/02601 2 1 4 Q 7 8 5 PCT/US93/06830
DescriPtion
Non-Native Liver Generation in an
Animal With ImPaired Native Liver Function,
bY Cell ImPlantation
Technical Field
The invention relates to non-native functioning liver
generation in animals with impaired native liver function, and
to (transgenic) animals harboring functioning non-native livers.
Backqround Art
There is a current notable shortage of donor livers. In
1990, illustratively, 2,656 liver transplants were performed, but
10 times more people died of liver disease. The treatment of
liver disease by the implantation of healthy liver cells has long
been envisioned as a potential solution to liver diseases.
Hepatocyte transplantation could be used to treat patients with
congenital hepatic enzyme deficiencies to replace liver lost
through disease (e.g. viral hepatitis, alcohol-related cirrhosis
or fibrosis), or provide a temporary support system where acute
liver dysfunction may be resolved by regeneration over a period
of time. Hepatocyte transplantation also offers the opportunity
to create animal models with human liver function. Such animals
would be very useful for the study of viral infections affecting
the liver (e.g. human hepatitis virus or cytomegalovirus
infection) and/or test the effectiveness and safety of treatment
or therapies for such infections. They would also be useful for
the study of chemical (e.g. alcohol) toxicity in the human liver
and chemical-induced cirrhosis and fibrosis and to test the
- effectiveness and safety of treatments or therapies for
chemical-induced liver damage.
Historically, several methods have been pursued in an
attempt to create an auxiliary liver. Transplantation of liver
fragments had been carried out as early as the 1930s. However,
in most cases the transplanted liver tissue either degenerated
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or disappeared within a few days or weeks.
Greater success in hepatocyte transplants have been observed
through the development of methods for isolating viable
hepatocytes. Among these methods, the "digestion-perfusion"
method of Barry and Friend (J. Cell Biol.,`(1969) 43:506-520),
block perfusion (Rees and Byard, In Vitro, (1981) 17:935-941),
and injection-digestion (Woods et al, Transplantation, (1982)
33:123-126) have produced good yields of viable mature
hepatocytes for injection. For a review see Gupta et al,
HePatoloqy, (1992) 15:156-162.
In addition to problems in hepatocyte isolation, the method
and site of implantation have been investigated for their effect
on transplantation. Direct infusion of hepatocyte suspensions
into the portal vein of rats has been investigated. However,
contrary to initial beliefs, the portal blood supply was not
found to be paramount to hepatocyte survival. Demonstrations
have been made for hepatocyte growth following intramuscular
injection, abdominal cavity injection, spleen, liver and kidney
inoculation, and injection into celiac or renal arteries. While
the above-mentioned methods have been able to produce small
amounts of viable implanted hepatocytes, the resulting hepatic
contribution to function has been relatively small and often
temporary.
Another currently investigated approach to liver transplant
resides in the implantation of new liver cells on scaffolds of
biodegradable polymers that are hoped to regenerate into masses
and take over at least some of the native liver's functions.
Although it has been reported that with this approach liver cells
on a matrix can survive and function in rats six months to a year
after transplantation, implanted cell survival is low, and, in
some animal trials, too much connective tissue grew into the
matrix, binding tightly to the polymer and forming scar tissue
preventing the liver cells from dispersing through the matrix
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("Technology Review~ July 1992, pp. 12, 13).
In a different vein, transgenic animal technology has
provided the means to create and analyze animal models of genetic
diseases affecting virtually any organ to which transgene
expression can be directed. Because of its involvement in many
diseases of medical importance, the liver has been a frequent
target for this type of analysis.
Among transgenes reported to be associated with liver
lesions are those encoding growth hormone, which alters
hepatocyte size and nuclear characteristics; oncogenes, which
induce hepatic tumors; the hepatitis B virus large envelope
polypeptide, which induces hepatocellular necrosis and carcinoma
formation in adults; transforming growth fact ~, which causes
both tumors and excessive liver growth; and a variant form of
~l-antitrypsin (AAT), which reproduces some characteristics of
AAT deficiency disease in humans, including neonatal and adult
hepatitis.
Because of the liver's large synthetic and secretory
capability, targeting transgene expression to this organ also
serves as a way to determine the systemic influence of
overproduction of biologically potent molecules. This approach
has previously been used to study the consequences of
inappropriate plasminogen activator expression in vivo by
introducing into mice a transgene bearing the urokinase-type
plasminogen activator (uPA) coding sequence fused to the albumin
enhancer/promoter (Heckel et al. Cell (19900 62:447-456).
Plasminogen activators catalyze the proteolytic cleavage and
activation of plasminogen to plasmin, which in turn degrades
fibrin clots (Collen, D. and Lijnen, H.R. (1987) Fibrinolysis and
the Control of Hematostasis. In the Molecular Basis of Blood
Diseases. G. Stamatoyannopoulos, A.W. Nienhuis, P. Leder, and
P.W. Majerus, eds. (Phila.: W.B. Saunders Co.), pp. 662-688).
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uPA also has been implicated in biological processes involving
tissue remodeling or destruction, including ovulation, m~mm~ry
gland involution, and metastasis (Dan0 et al., Adv. Cancer Res.
(1985) 44:149-265; Collen and Lijnen, (1987) supra; Saksela and
Rifkin, Annu. Rev. Cell Biol. (1988) 4: 93-126) .
In many transgenic founder mice the albumin-urokinase type
plasminogen activator (Alb-uPA) construct directed high-level uPA
expression to hepatocytes, resulting in elevated plasma uPA and
fatal hemorrhaging in newborns. Two lines of Alb-uPA transgenic
mice were established from surviving founder mice that expressed
lower levels of uPA. In these lines, about half of the
transgenic neonates bled into either the intestine or abdomen and
died within four days after birth, whereas the remaining
transgenic offspring appeared normal and survived. Both bleeding
and non-bleeding transgenic neonates displayed marked
hypofibrinogenemia and unclottable blood, and it was concluded
that any injury sufficient to initiate bleeding was rapidly fatal
in affected mice (Heckel et al. (199O), supra).
A general expectation in experiments of this type is that
transgene expression will be stable over time in targeted cells.
Unexpectedly, surviving mice in both of the Alb-uPA lines showed
a gradual decrease in the level of plasma uPA activity
accompanied by a restoration of clotting function that was
complete between 1 and 2 months of age (Heckel et al., (199O)
supra) . This phenomenon has been explained by a report that uPA
is cytotoxic for hepatocytes and that inactivation of transgene
expression by DNA rearrangement in isolated hepatocytes in
Alb-uPA mice is followed by repopulation of the entire liver by
cells that no longer produce uPA (Sandgren et al, Cell (1991)
66:245-256) . Thus, Alb-uPA expression provides a transgenic
mouse in which endogenous transgene-expressing hepatocytes have
a selected disadvantage relative to hepatocytes (native or
non-native) that are not expressing the transgene. In addition,
the livers in these mice provide an environment for growth of
WO94/02601 2 1 4 07 8 S PCT/US93/06830
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hepatocytes (native or non-native) that are not expressing the
transgene. These cells grow out at the expense of transgene-
expressing cells and the result is livers with apparently normal
architecture.
Disclosure of the Invention
Despite progress made in the implantation and development
of mature hepatocytes, present technology has not made possible
the generation of a (functioning) non-native liver into an
animal, particularly one with defective liver function. In
humans, such technology would be useful in liver disease therapy
and/or treatment. In non-human animals such technology would
have considerable value, e.g. to provide models of (diseased)
human liver function useful to study potential treatment and/or
therapies for human liver disease.
Accordingly, one object of the invention is to provide a
method of correcting defective liver function in an animal host
by implanting particular non-native cells (or corresponding
tissue) into the liver region of a host animal with impaired
native liver function to thereby generate a functioning
non-native liver in the host and correct the impaired liver
function. Another object of the present invention is to provide
transgenic, non-human animals (e.g. rodents such as mice)
harboring a transgene that encodes a product that is
disadvantageous, but not lethal, to native liver cells and thus
impairs native liver function. These transgenic, non-human
animals are useful as a host system for non-native (e.g. human)
liver. Another object of the present invention is to provide
non-human host animals that can both maintain and expand a
fully-functioning non-native (e.g. human) liver tissue useful for
(l) modeling liver disease, (2) liver tissue banking (e.g.
maintenance and expansion of liver tissue for later analysis and
re-implantation back into donors), (3) testing pharmaceuticals
for human liver toxicity in vivo, and/or (4) genetic manipulation
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prior to re-implantation (i.e., hepatocyte-directed gene
therapy). Another object of the invention is to provide a method
of generating a functioning non-native liver in a host animal by
implanting non-native liver cells (or tissue) into a host animal
with a genetically established defective liYer function. Another
object of the invention is to provide non-human animals harboring
a functioning non-native liver. Another object of the invention
is to provide a novel method for maintaining full-differentiated,
full-functioning donor (e.g. human) hepatocytes in an in vivo
setting for genetic manipulation prior to return to the donors.
The genetic manipulation could include, for example, introducing
expression vectors that direct the production of medically
important proteins in the transplant recipient.
It has been discovered by the inventors that the above
objects of the invention and other objects which will become
apparent from the description of the invention given hereinbelow
are satisfied by implanting non-native cells (e.g, fetal,
neonatal or adult, non-hematopoietic liver stem, progenitor or
mature cells; fetal, stem, progenitor or adult bile duct cells;
endodermal (e.g. pancreas or gut) cellsi (cultured) totipotent
stem cells; or cultured animal cells), or corresponding tissue,
into the liver region of a host animal having impaired native
liver function, such that the implanted cells (or tissue)
colonize the host animal and develop therein a functioning
non-native liver.
Best Mode for Carryina Out the Invention
Any animal having impaired native liver function may be used
in accordance with the invention as a suitable host animal.
Suitable host animals therefore include fish, fowl (e.g.
chickens, ducks, geese, turkeys, etc.), or m~mm~l S such as
rodents (e.g. mice or rats), guinea pigs, pigs, dogs, cats,
rabbits, goats, sheep, horses, ruminants such as cows, monkeys,
other non-human primates, as well as humans.
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Impairment of native liver function in the host animal may
have occurred accidentally, e.g. through disease, such as
cirrhosis, hepatitis, fibrosis or a genetic liver deficiency
disease such as deficiencies in Factor IX, Factor VIII, LDL
receptor, or one of varied metabolic enzymes (e.g. phenylalanine
hydroxylase), or any of the other hundreds of known metabolic
diseases that affect the liver.
In a preferred embodiment, the invention is applied as
therapy and/or treatment to such animals, including humans,
suffering from liver failure. In this context, the invention is
used as an alternative to liver transplant to restore liver
function.
In another embodiment, a non-native liver is generated in
a non-human animal in which liver insufficiency has been induced
to obtain an animal model system with a functioning non-native
(e.g. human or other animal) liver. In these non-human animals,
impairment of native liver function may be purposefully achieved
by compromising the native hepatocyte genetically so that they
are at a selective disadvantage towards implanted non-native
cells (e.g. hepatocytes).
In accordance with the invention, during normal turnover of
hepatocytes, the implanted non-native hepatocytes have a
proliferative advantage over the accidentally or purposely
disadvantaged native hepatocytes and ultimately replace all of
the native hepatocytes in the host. This provides a host
harboring substantially few, if any native hepatocytes, but a
functioning, substantially non-native, liver generated from the
implanted non-native cells. (The liver generated is
substantially non-native insofar as it is likely to comprise some
non-hepatocyte cells found in the native liver and perhaps some
native hepatocytes.)
Genetic inducement of liver insufficiency in a non-human
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mammal may be achieved in accordance with this embodiment of the
present invention with a transgenic, non-human animal harboring
a transgene which encodes a product that is disadvantageous, but
not immediately lethal, to native liver cells. Generally, any
deleterious transgene product may be used that impairs cellular
function to the extent that hepatocytes expressing the gene have
a distinct growth disadvantage relative to cells that do not
express a transgene (e.g. the implanted non-native hepatocytes).
Examples of such transgene products include any type of
plasminogen activator, such as an urokinase-type or tissue-type
plasminogen activator, or bacterial plasminogen activators (e.g.
strptokinase) or toxins such as an attenuated diphtheria toxin.
Other examples of transgene products useable in accordance with
this embodiment of the present invention include agents that
adversely affect the metabolism or growth potential of the native
liver cells. Illustrative agents are agents which compromise the
ability of the native liver cells to transmit growth factor
signals (e.g. with dominant and negative transgenes affecting
steps in signal transduction pathway) or by producing mutant
proteins that clog circulatory pathway(s).
In an illustrative genetic inducement of liver failure, the
expression of high levels of urokinase-type plasminogen activator
(uPA) has been reported by the inventors to cause impaired liver
function in m~mm~l S . Sandqren et al in Cell (1991) 99:245-256.
Surviving transgenic animals harboring Alb-uPA are born with a
liver consisting of smooth, pale to nearly white tissue, "white
liver," within which develop multiple reddish nodules which
expand in size with age. The white areas represent original
transgenic liver tissue while the red areas represent post-natal
changes in some aspects of liver function and structure which
give rise to red nodular growth. The white tissue samples
express high levels of uPA mRNA whereas all samples of the red
nodules lack detectable transgene expression.
W094/02601 2 1 4 U78 5 PCT/US93/06830
This white liver tissue contains small hepatocytes with
altered rough endoplasmic reticulum morphology but is neither
necrotic or fibrotic. Expression of the uPA transgene appears
to be deleterious to hepatocytes, but not immediately lethal.
This kind of genetic impairment of native liver function provides
a suitable format for establishing regenerative liver outgrowths
from either donor animals (e.g. humans) or endogenous liver cells
that spontaneously cease uPA transgene expression through DNA
recombination and loss of all functional copies of the transgene
tandem array.
In the later case, heterozygous Alb-uPA mice frequently
develop regenerative liver nodules with normal reddish liver
color that grow out at expense of surrounding "white" liver
tissue and eventually reconstitute the liver. These "red" liver
nodules are clonal outgrowth of single hepatocytes based on
analysis of the transgene remnants left behind following
transgene recombination. Hepatocytes that have deleted all
functional transgenes, and lack detectable transgene express,
acquire a selected advantage and quickly expand and replace the
transgene-expressing hepatocytes in the surrounding "white
tissue~. An embodiment that is an extension of these
observations is that non-native (e.g. human) liver cells can be
transplanted into these mice (or their functional equivalent)
and, as a result of their inherent growth advantage over native,
transgene-expressing liver cells, they grow out to reconstitute
the liver at the expense of endogenous liver cells which do die
or disappear.
According to this general genetic inducement method, a
transgene that encodes a product which is disadvantageous, but
not immediately lethal, to the liver cells of the host animal is
constructed. The resulting fusion construct is inserted using
known techniques into fertilized eggs followed by implantation
into pregnant or pseudopregnant females. The progeny are then
bred to produce offspring which express the construct, and which
WO94/02601 PCT/US93/06830
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have impaired native liver function.
A variety of different non-native cells (or corresponding
tissue) may be used for implantation into t~he host in accordance
with the invention. These cells (or tissue) may be fetal,
neonatal and/or adult stem, pragenitor and/or mature
non-hematopoietic liver cells. Preferably non-hematopoietic
liver progenitor cells and more preferably non-hematopoietic
liver stem cells are used. In this context, hepatocytes, which
constitute about 60~ of the mammalian liver, may be used as the
liver cells.
The non-native hepatocytic stem cells which may be used in
the invention are distinct from fetal hematopoietic stem cells.
Fetal hematopoietic stem cells are precursors to the
hematopoietic system, and have previously been used to generate
a non-native hematopoietic system in a host.
Other non-native cells (or tissue) which may be used in
accordance with the invention include (i) stem, progenitor and/or
mature bile duct cells, (ii) endodermal tissue cells such as
pancreas or gut cells, (iii) optionally cultured totipotent stem
cells including (ES) cells or embryonic carcinoma (EC) cells,
and/or (iv) cultured animal cells.
In a particularly preferred embodiment, totipotent animal
stem cells are first cultured under conditions which control
their differentiation, such as by using particular growth factors
in the cell culture medium, and these cultured cells are then
used to colonize the host animal. Totipotent animal stem cells
can also (alternatively) be first treated or cultured with growth
factors that enhance survival, promote replication, and/or
selectively enrich for stem cells.
Animal cells (or tissue) from other sources, including but
not limited to embryonic stem cells or fetal endoderm, can also
WO94/02601 2 1 4 0 7 8 S PCT/US93/06830
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be treated by growth factors or other substances to bring about
differentiation of a particular cell lineage with characteristics
of hepatic stem cells. Illustratively, embryonic stem cells or
embryonal carcinoma cells can be stimulated to differentiate by
retinoic acid or another inducer. Subsequent differentiation in
culture along the endodermal or hepatocyte cell lineage can be
achieved by stimulation with growth factors and/or selection,
using known techniques and materials.
In an embodiment of the present invention, the non-native
cells (or tissue) may by genetically engineered (using known
techniques) to produce, in vivo in the host, a physiologically
effective amount of one or more medically relevant protein and
then transplanted in accordance with the present invention. This
technique can be used to improve hepatocyte function in an
animal, e.g, by producing Factor IX, LDL receptors, phenylalanine
hydroxylase, etc. or to produce proteins of systemic value, e.g.
Factor VIII, adenosine de~m~n~se, or peptide hormones.
Thus, non-native cells (or tissue) to be implanted may be
isolated from a donor animal, as noted below, and optionally
cultured, using known techniques. For example, in accordance
with an embodiment of the invention, non-hematopoietic liver
stem, progenitor and/or mature cells are first isolated from the
terminal or transitional bile ductiles of a mature donor animal
other than the host animal, using known techniques. In another
illustrative embodiment, hepatic cells may be isolated from the
donor animal using known procedures, e.g. by extraction through
a biopsy needle. Alternatively, hepatic cells may be isolated
from the donor animal by collagenase perfusion of the portal
vein.1
The donor animal used in accordance with the invention may
1See, e.g. , Soda et al, Blood (1984) 63, 270-276, or
Claunig et al, In Vitro (1981) 17, 913-925.
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2140~ 8S -12-
be an animal of the same species or of a different species as
compared to the host animal. Generally it is a different
individual. The donor animal may be any animal harboring a
normal functioning liver or an animal with a congenital or
squired liver disease. In a preferred embodiment the donor
animal is a human.
The non-native cells (or tissue) are then introduced into
the host organism in a manner which provides for implantation of
a sufficient number of the non-native cells into the liver region
of the host animal to permit their development into a non-native
liver. The present invention does not require the use of a
polymeric matrix for supporting the implanted cells. Once
implanted into the liver region of the host animal, the injected
cells (or tissue) are subjected therein, by virtue of their
location, to a variety of native biological signals to which the
native liver is normally subjected and which provide for the
growth and maintenance of implanted non-native liver cells in the
host.
Thus, cells from a variety of sources may be used in
accordance with the invention, since such cells are comprised of
a mixture of cells of different degree of development (i.e.,
stem, progenitor and mature cells). These mixtures contain cells
having a sufficiently primitive degree of development so that,
when subjected to the biological environment of the host~s liver
area, these cells will establish a functioning non-native liver.
Illustratively, other cells, particularly of endodermal
origin, may be used to form a new liver. For example, Scarpelli
et al in Laboratory Investiqation (1990) 62:552 reported
identifying cells in the pancreas of hamsters, treated with
carcinogens, which have the appearance of hepatocytes. These
cells and others may form functional hepatocytes when placed in
the appropriate environment (e.g. a failing liver) in accordance
with the present invention.
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Implantation into the host animal's liver area may be
achieved using different techniques. Illustratively, a
suspension of from lx103 to lx107, preferably lx104 to lx106,
non-native cells, optionally together with additional adjuvants
to promote the transplantation of the non-native cells (i.e.,
substances which are known to enhance non-native cell survival
and/or division2), are administered (e.g. injected through a
syringe needle) into any of several different sites where
colonization of the non-native cells may be observed.
Suitable sites include the abdominal cavity, muscle tissue,
kidney, pancreas, celiac artery, fat pads and/or subcutaneous
area of the host animal. Preferred sites of administration in
accordance with the invention, and which are preferably
effectuated by injection, include the portal vein, the spleen,
directly into the native liver, and the umbilical vein of the
fetal host which leads to the fetal liver.
In accordance with the invention, if the host and donor are
two different animals steps are taken prior to implantation to
suppress potential host immune rejection of the implanted
non-native cells. This may be achieved in a variety of ways,
such as by treating the host animal with cyclosporin or any other
material known to suppress the host's immunological system, e.g.
agents that are known to selectively destroy subgroups of the
immune cell population of the host, such as T-cell destroying
antibodies. Alternatively, tolerance in the host can be induced
by transferring non-native cells to the thymus of the host before
implantation (see, Rosselt et al, Science (1990) 249:1293-1295),
or cells from an animal of the same strain as the host can be
used. Still further, an immunodeficient host can be used, such
as inbred strains of an animal (e.g. SCID mice or nude mice) bred
to act as recipients of non-native cells.
2For example, cells can be introduced together with growth
factors or other substances that enhance survival, implantation,
and for growth.
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In an illustrative and preferred embodiment, the recipient
for the non-native cells is a transgenic mouse with defective
liver function, and human non-hematopoietic liver cells are used
to generate the non-native liver. A suitable transgenic mouse
is described by Sandgren et al in Cell, (l99l) 66:245-256, which
is hereby incorporated by reference. The transgenic mouse
described in this publication of the inventors exhibits a high
expression of albumin-urokinase-type plasminogen activator
(Alb-uPA) fusion construct. This mouse with impaired liver
function is a good recipient for human non-hematopoietic liver
cell implantation (but mice with liver impairment resulting from
other causes may be used).
The transgenic mouse with impaired liver function is
injected with non-native, preferably human non-hematopoietic
liver cells such that the injected human hepatocytic stem cells
colonize the native liver at the expense of the endogenous "white
tissue~ of the transgenic mouse. The human liver cells
proliferate to form nodules which ultimately reconstitute the
entire native liver of the host with human hepatocytes and
establish human liver function. This includes the replacement
of most mouse plasma proteins with their corresponding human
plasma proteins.
The resulting mouse or any other non-human host produced in
accordance with the invention, is well suited for (l) modeling
study of human liver function, including screening pharmaceutical
agents for treating human liver disease, (2) modeling human
congenital diseases affecting the liver (including those in which
the cause is unknown) by implanting patient liver cells into host
mice, (3) liver cell banking (e.g. maintenance and expansion of
liver tissue for later analysis or re-implantation into the
donor), (4) genetic manipulation of full-functioning,
full-differentiated hepatocytes for later re-implantation into
donors (i.e., hepatocyte-directed gene therapy such as hepatitis
virus resistant human liver cells that express antisense against
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the virus), and/or (5) modeling human liver for analysis of
therapeutic agents that can promote liver regeneration.
More particularly, the present invention provides:
(i) Methods for maintaining and generating fully-
functional, full-differentiated liver tissue for future analysis
and therapeutic uses;
(ii) Therapeutic uses including: (a) re-implantation of
healthy liver tissue into donor patients once an infectious agent
or diseased tissue has been eliminated, and (b) genetic
modification of donor cells and subsequent re-implantation into
donor patients (e.g. hepatocyte-directed gene therapy);
(iii) Animal model systems with functioning, non-native
liver (e.g. human hepatocytes) for the study of viral infections
(e.g. human hepatitis virus or cytomegalovirus infection) and to
test the effectiveness and safety of treatments or therapies for
such infections;
(iv) Animal model systems with functioning, non-native
(e.g. human) liver for the study of chemical (e.g. alcohol)
toxicity in the liver and chemical-induced cirrhosis and fibrosis
and to test the effectiveness and safety of treatments or
therapies for chemical-induced liver damage;
(v) Methods for generating non-human animal models of
congenital human liver disorders (even when the cause is not
known) by reconstitution of host animal livers with human liver
hepatocytes collected from patients diagnosed with liver disease;
(vi) Animal model systems with functioning, non-native
(e.g. human) liver which models congenital human liver disorders
(e.g. , glycogen storage diseases, a,-antitrypsin deficiency,
coagulation factor disorders and deficiencies) for use in testing
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the effectiveness and safety of treatments and therapies;
(vii) Methods for generating non-human animals in which the
liver-derived plasma proteins (which comprise >90~ of the total
plasma proteins) are derived from transplanted, non-native (e.g.
human) liver cells;
(viii) Animal systems with a functioning, non-native (e.g.
human) liver in which to generate and study normal or abnormal
donor-liver derived plasma proteinsi
(ix) Animal systems with a functioning, non-native (e.g.
human) liver for testing the effectiveness and safety of
treatments or therapies for disorders affecting donor
liver-derived plasma proteins; and
(x) Systems for generating in large quantities of
recombinant plasma proteins for therapeutic or diagnostic use.
Hepatocytes are equipped to assemble, and appropriately modify,
copious amounts of plasma proteins; thus, transplanted
hepatocytes, or their genetically engineered derivatives, may be
used to generate valuable donor proteins or other derived
proteins.
* * * * *
Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings.
It is therefore to be understood that within the scope of the
appended claims, the invention may be practiced otherwise than
as specifically described herein.
