U.S. patent application number 12/180851 was filed with the patent office on 2009-02-05 for human albumin animal models for drug evaluation, toxicology and immunogenicity studies.
Invention is credited to Daniel C. CARTER.
Application Number | 20090038020 12/180851 |
Document ID | / |
Family ID | 23149151 |
Filed Date | 2009-02-05 |
United States Patent
Application |
20090038020 |
Kind Code |
A1 |
CARTER; Daniel C. |
February 5, 2009 |
HUMAN ALBUMIN ANIMAL MODELS FOR DRUG EVALUATION, TOXICOLOGY AND
IMMUNOGENICITY STUDIES
Abstract
An animal model is provided which is genetically engineered to
express human serum albumin, and such animals may be advantageously
used in assessing drugs, vaccines or other therapeutic compounds
that may be used in humans. In addition, an animal model is
provided which does not manufacture its own albumin and which has
been injected with human serum albumin. Through the use of these
animal models, drugs and other chemicals can be more accurately
assessed in physiological environments that reflect the conditions
to be expected in humans, and such models will be useful in
assessing new drugs and evaluating toxic substances for potential
dangers as carcinogens, mutagens, etc. Other applications include
evaluating immunological properties of various albumin-engineered
proteins which might be administered to humans as therapeutics or
vaccines, and research of disease states, such as genetic diseases,
to provide further insight in treating these diseases.
Inventors: |
CARTER; Daniel C.;
(Huntsville, AL) |
Correspondence
Address: |
STITES & HARBISON PLLC
1199 NORTH FAIRFAX STREET, SUITE 900
ALEXANDRIA
VA
22314
US
|
Family ID: |
23149151 |
Appl. No.: |
12/180851 |
Filed: |
July 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11232989 |
Sep 23, 2005 |
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12180851 |
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10171688 |
Jun 17, 2002 |
6949691 |
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11232989 |
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60298124 |
Jun 15, 2001 |
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Current U.S.
Class: |
800/3 ;
800/14 |
Current CPC
Class: |
A01K 2207/15 20130101;
A01K 67/0278 20130101; C07K 14/765 20130101; A01K 2267/03 20130101;
C12N 15/8509 20130101; A01K 2217/00 20130101; A01K 2227/105
20130101; A61P 43/00 20180101; A01K 2217/05 20130101 |
Class at
Publication: |
800/3 ;
800/14 |
International
Class: |
G01N 33/00 20060101
G01N033/00; A01K 67/00 20060101 A01K067/00 |
Claims
1. A transgenic rat whose genome comprises a nucleic acid encoding
human serum albumin that is expressed in the rat's bloodstream,
wherein the gene coding for the native rat serum albumin is
replaced by the nucleic acid coding for human serum albumin such
that the rat does not express its own natural serum albumin.
2. The transgenic rat according to claim 1 wherein the rat has been
mutated so that it does not express its own natural serum
albumin.
3. The transgenic rat according to claim 1 wherein the rat is an
NAR rat.
4. The transgenic rate according to claim 1 wherein said rat has
had its gene for serum albumin knocked out.
5. A method of testing the efficacy, carcinogenicity,
immunogenicity or toxicity of an agent designed to be administered
internally to a human selected from the group consisting of
vaccines, drugs and other therapeutic compounds comprising the
steps of administering internally said agent into the transgenic
rat according to claim 1 and determining the efficacy,
carcinogenicity, immunogenicity or toxicity of said agent in said
transgenic rat.
6. The method according to claim 5 wherein the testing is to
evaluate the efficacy of a drug or other therapeutic compound.
7. The method according to claim 5 wherein the testing is to
evaluate the carcinogenicity or toxigenicity of a drug or other
therapeutic compound.
8. The method according to claim 5 wherein the testing is to
evaluate the immunogenicity or other immunological properties of a
vaccine.
9. A transgenic NAR rat whose genome comprises nucleic acid coding
for human serum albumin which is expressed in the animal's
bloodstream, wherein the rat does not express its own natural serum
albumin.
10. A transgenic rat whose genome comprises a nucleic acid encoding
human serum albumin that is expressed in the rat's bloodstream.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 11/232,989, filed Sep. 23, 2005, which was a divisional
application of U.S. Ser. No. 10/171,688, filed Jun. 17, 2002, now
U.S. Pat. No. 6,949,691, issued Sep. 27, 2005, which claims the
benefit of U.S. Provisional Application Ser. No. 60/298,124, filed
Jun. 15, 2001. All of said applications are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] This invention relates in general to an animal model with
serum attributes which have similar or identical properties and
pharmokinetics with the human blood system in order to improve the
assessment and development of new therapeutics and vaccines and to
provide more reliable systems for identifying chemicals or drugs
that may be carcinogenic or toxigenic to humans, and more
specifically relates to transgenic animals and other animal models
which contain and/or express human serum albumin and which thus can
be used in methods of drug evaluation, toxicology and
immunogenicity so as to provide a far more accurate picture of how
drugs and chemicals will react in humans than would be possible
through the conventional testing of animals that do not express
human serum albumin.
BACKGROUND OF THE INVENTION
[0003] The serum albumins belong to a multigene family of proteins
that includes alpha-fetoprotein and human group-specific component,
also known as vitamin-D binding protein. The members of this
multigene family are typically comprised of relatively large
multi-domain proteins, and the serum albumins are the major soluble
proteins of the circulatory system and contribute to many vital
physiological processes. Serum albumin generally comprises about
50% of the total blood component by dry weight, and as such is
responsible for roughly 80% of the maintenance of colloid osmotic
blood pressure and is chiefly responsible for controlling the
physiological pH of blood.
[0004] The albumins and their related blood proteins also play an
extremely important role in the transport, distribution and
metabolism of many endogenous and exogenous ligands in the human
body, including a variety of chemically diverse molecules including
fatty acids, amino acids, steroids, calcium, metals such as copper
and zinc, and various pharmaceutical agents. The albumin family of
molecules are generally thought to facilitate transfer of many of
these ligands across organ-circulatory interfaces such as the
liver, intestines, kidneys and the brain, and studies have
suggested the existence of an albumin cell surface receptor. See,
e.g., Schnitzer et al., P.N.A.S. 85:6773 (1988). The albumins are
thus involved in a wide range of circulatory and metabolic
functions.
[0005] Human serum albumin (HSA) is a protein of about 66,500 kD
and is comprised of 585 amino acids including at least 17
disulphide bridges. As with many of the members of the albumin
family, human serum albumin plays an extremely important role in
human physiology and is located in virtually every human tissue and
bodily secretion. Human serum albumin is the major protein of the
circulatory system and as such is involved in the attachment,
distribution and metabolism of all known pharmaceuticals because of
its particular binding affinities to these chemicals. The atomic
structure and particular details regarding the binding affinities
of albumin and the specific regions primarily responsible for those
binding properties have been previously determined as set forth,
e.g., in U.S. Ser. No. 08/448,196, filed May 25, 1993, now U.S.
Pat. No. 5,780,594 and U.S. Ser. No. 08/984,176, filed Dec. 3,
1997, now U.S. Pat. No. 5,948,609, both of which are incorporated
herein by reference. Other articles or references of relevance with
regard to human serum albumin include Carter et al., Advances in
Protein Chemistry, 45:153-203 (1994); Peters, Jr., "All About
Albumin", Academic Press (1995); Camerman et al., Can J. Chem.,
54:1309-1316 (1976); Lau et al., J. Biol. Chem., 249:5878-5884
(1974); Callan et al., Res. Commun. Chem. Pathol. Pharmacol.,
5:459-472 (1973); and Nieboer et al., Br. J. Ind. Med., 41:56-63
(1984); and all of these references are incorporated by reference
as well.
[0006] It is also widely known and understood that the amino acid
sequences in the different animals vary from humans in differing
degrees, from essentially 98% homology for chimpanzees to a more
typical value of approximately 60% for other animals such as dogs,
mice, rats, etc. While data has shown the overall conservation of
the three-dimensional structure of albumin from other species, the
specific residues involved in the ligand binding chemistry are
distinctly different and account for sometimes quite different
pharmokinetics and toxicity involving drug experiments with animal
models. As a result, the literature is replete with examples of
drugs which appeared to be extremely effective in animal testing
only to show extremely disappointing results when used in human
trials.
[0007] There is thus a significant need to develop new animal
models which can more accurately be utilized in toxicological and
pharmokinetic studies so as to more accurately reflect how a
particular drug or other chemical will work in humans.
SUMMARY OF THE INVENTION
[0008] Accordingly, it is thus an object of the present invention
to create animal models which supplement or replace the natural
albumin sequence with a typical human serum albumin sequence and
which thus will be far more effective in providing an accurate
assessment of how drugs or other chemicals will react in
humans.
[0009] It is another object of the present invention to create
transgenic animals which contain and/or express human serum albumin
and which will thus be far more useful in methods of drug
evaluation, toxicology and immunogenicity and provide a far more
accurate picture of how drugs, chemicals and vaccines will react in
humans than would be possible through conventional testing of
animals that do not express human albumin.
[0010] It is still further an object of the present invention to
create a variety of transgenic animals from a variety of species
which will contain and express human serum albumin and which will
thus be far more useful in assessing the safety and efficacy of new
drugs, vaccines, or other medicaments.
[0011] It is yet another object of the present invention to provide
even further animal models for assessing the safety and efficacy of
materials such as drugs or vaccines which are intended to be
administered internally to humans, said models which are either
natural mutants which do not express their own serum albumin or
mammals which have had the gene for serum albumin knocked out, and
such models can be injected with human serum albumin which is
retained in these mammals and thus provides an animal model for
assessing drugs or vaccines in humans more accurately than in
animal models which do not contain or express human serum
albumin.
[0012] It is still further an object to provide hybrid non-human
mammals which are transfected with the gene for human serum albumin
yet maintain their own expression of serum albumin, and thus such
hybrid animals may be used in methods of testing or assessing
internal medicines, or in research involving administration of such
medicines in cases with a given percentage of human serum albumin
versus a given percentage of a serum albumin from a non-human
mammal.
[0013] It is even further an object of the present invention to
provide a method for assessing human drugs or vaccines, or other
chemicals which may be utilized in compositions taken by or applied
to humans, by which the drug, vaccine or chemical can be assessed
in a manner more likely to be consistent with the way that these
materials will be handled by the human body.
[0014] It is yet a further object of the present invention to
provide animal models which reflect the various genetic diseases
and disease states and which also incorporate and express a gene
for human serum albumin so as to be useful in methods of assessing
drugs utilized to treat or prevent genetic diseases or disease
conditions caused by other pathogens.
[0015] These and other objects are achieved by virtue of the
present invention which provides animal models wherein the animal
contains human serum albumin in its bloodstream, either through
genetic engineering to transfect a gene coding for human serum
albumin, either in conjunction with or as an alternate to the
animal's own genes for producing albumin, or through the use of
animals which do not produce their own native albumin and which
have been injected with human serum albumin which is retained in
their bloodstream. The invention also provides methods of preparing
such animals and of using such animals in the assessment of drugs,
vaccines, chemicals or any other materials that may be used on
humans. Having distributions of the drugs in the circulatory system
which directly reflect the conditions to be expected in humans is
of tremendous value to the pharmaceutical industry and also has
great application in the evaluation of various toxic substances for
potential dangers as carcinogens, mutagens, etc. In addition, there
are numerous applications involving the evaluations of
immunogenicity or other immunological properties of various
albumin-engineered proteins which might be administered to humans
as therapeutics or vaccines, and these will also now be able to be
assessed in animal models for closely following the human blood
system.
[0016] The animal models of the present invention, including
transgenic animals expressing human serum albumin as well as other
models such as mutant rats which do not produce their own albumin
and which can be injected with human serum albumin, can thus be
used advantageously so as to assess drugs or other internal
medicines for their effect in the human bloodstream and/or their
reactions to a more normal human setting, namely a bloodstream
having human serum albumin therein. In addition, it is also
possible to employ the transgenic animals in accordance with the
present invention for the large-scale production of human serum
albumin when such production is desired.
[0017] These and other features of the present invention as set
forth in, or will become obvious from, the detailed description of
the preferred embodiments provided hereinbelow.
BRIEF DESCRIPTION OF THE DRAWING FIGURES
[0018] FIG. 1 is a chromatograph showing the results of human serum
albumin testing involving animal model ODNAR10 in accordance with
the invention, including lane 1, the molecular weight marker, lane
2, a Sprague-Dawley (SD) control rat, lane 3, blank, lane 4, NAR
control rat, lane 5, serum from female NAR rat 22, lane 6, serum
from female NAR rat 23, lane 7, serum from fee NAR rat 24, lane 8,
serum from male NAR rat 30, lane 9, blank, and lane 10, blood
sample from ODNAR10 after fifth bleed.
[0019] FIG. 2 is a chromatograph showing the results of human serum
albumin testing involving animal model ODNAR14 in accordance with
the invention, including lane 1, the molecular weight marker (New
England Biolabs, Prestained Protein Marker, Broad Range #P7708S,
premixed, Lot 25), lane 2, SD control rat, lane 3, blank, lane 4,
pre-HSA injection bleed of ODNAR14, lane 5, blank, lane 6, ODNAR14
after 4.sup.th post-injection bleed, lane 7, ODNAR14 after 3.sup.rd
post-injection bleed, lane 8, ODNAR14 after 2.sup.nd post-injection
bleed, lane 9, ODNAR14 after 1.sup.st post-injection bleed, lane
10, blank.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0020] In accordance with the present invention, animal models are
provided which contain and/or express human serum albumin either in
conjunction with, or as an alternative to, the animal's own genes
for producing albumin. The amino acid and nucleic acid sequences
for human serum albumin are known and have been disclosed, e.g., in
U.S. Pat. No. 5,780,594 (Carter et al.) and in the journal articles
Hawkins et al., Gene 19 (1):55-8 (1982) and Lawn et al., Nucleic
Acids Res. 9 (22):6103-114 (1981), all herein incorporated by
reference. In one preferred mode of the invention, the transgenic
animal models of the present invention are constructed via genetic
engineering of an appropriate animal so that genes coding for human
serum albumin are incorporated into the genome of the animal which
is then able to express human serum albumin. In one embodiment,
this genetically engineered animal may be constructed through the
introduction of genes coding for human serum albumin without
knocking out the animal's own genes for albumin. In these "hybrid"
animal models, the animals will thus have a range of percentages of
human serum albumin and of its own natural albumin in its plasma,
e.g., 50% human serum albumin and 50% animal serum albumin,
depending on the nature and extent of the introduced human serum
albumin genes. These hybrid mice are particularly suited for
immunological studies since they will express both types of albumin
and may not undergo an immunological reaction to either form of
albumin.
[0021] However, in another embodiment of the invention, an animal
model for drug testing and testing of other materials in
environments that will more closely approach human conditions is
prepared by introducing a gene for human serum albumin while
eliminating the animal's own genes for producing albumin. This type
of animal model, resulting in a "knock-out" and "knock-in" mouse,
will most closely approach the physiology of the human circulatory
system because the animal model in this case will have all of its
blood plasma dominated by human serum albumin and will not produce
the animal's own natural albumin. As described further below, in
the preferred animal model of the invention, it is highly desirable
to perform a "knock in" process simultaneously with the procedure
wherein the gene coding for the animal's native albumin is knocked
out and the gene for human serum albumin is "knocked in" at the
same location as the animal's own gene for serum albumin. This is
particularly desirable because the inserted gene for human serum
albumin will be at the most appropriate location in the genome to
interact with the other sequences involved in the operation and
expression of the albumin gene, and it is thus more likely that the
functioning of the animal and the expression of the albumin will be
carried out more normally if the human gene for albumin is inserted
at the animal's normal location for the albumin gene.
[0022] In accordance with the present invention, any animal that is
currently used for lab testing of materials such as drugs, vaccines
or other chemical substances, may be used as the animal model for
the present invention. In the typical case, the animals used will
be non-human mammals, including small mammals such as rabbits,
mice, rats, guinea pigs and other rodents. However, larger mammals,
such as dogs, are also used in lab testing and can be made
transgenic so as to express human serum albumin. In addition, other
mammals that have commonly been subject to transgenic processes,
such as pigs, sheep, calves and cows, may also be made into
transgenic animal models in accordance with the invention if so
desired for particular testing.
[0023] In this regard, it is contemplated that in addition to being
useful as animal models in accordance with the invention,
transgenic animals which are constructed in accordance with the
invention may be usable in the large scale production of human
serum albumin. In this aspect of the invention, it is generally
contemplated that those animals which may normally be used for
large-scale production of a transgenic products, particularly cows,
pigs, sheep or goats, may be utilized which will express human
serum albumin in their bloodstream which can then be isolated and
purified for use in human applications. In such a case, it would be
preferred if the transgenic animal of the invention is one wherein
the genetic material coding for the animal's own native albumin is
knocked out so that the animal only produces one kind of serum
albumin, namely human, which will assist in the isolation and
purification of the albumin obtained from transgenic animals
expressing human serum albumin in their bloodstream.
[0024] As would be recognized by one of ordinary skill in this art,
transgenic animals which contain and express human serum albumin
can be manufactured in a number of suitable ways which are now well
known in the art. For example, the methods of making transgenic
mammals such as disclosed in U.S. Pat. Nos. 4,736,866 (Leder et
al.) and 4,873,191 (Wagner), both incorporated herein by reference,
may be utilized in order to produce the transgenic animal models of
the present invention. In addition, numerous other methods of
genetically engineering animals so as to insert a particular gene
or to express an inserted gene are well known in the art and may be
used to produce the transgenic animal models of the present
invention.
[0025] In the preferred process of the present invention, the
nucleic acids coding for human serum albumin may be added to the
genome of a host animal by means of P-element derived vectors as
disclosed in U.S. Pat. No. 6,291,243, incorporated herein by
reference. In this process, the introduction of exogenous genetic
material coding for human serum albumin is achieved through the
insertion of P element-derived vectors, which include a pair of P
element transposase recognized insertion sequences. In the
preferred process, the desired albumin gene may be transferred into
the host mammal, e.g., through microinjection into mouse
oocyte/embryo nuclei to create a germ line transfer of genetic
material.
[0026] As indicated above, an animal model in accordance with the
preferred embodiment of the invention will have the gene coding for
human serum albumin inserted into its genome, and this gene will
include all necessary regions, including operons, promoters, etc.,
to ensure that the gene for human serum albumin will be expressed
in the animal model. In the particularly preferred embodiment, the
transgenic animal model containing and expressing human serum
albumin will be an animal which does not express its own serum
albumin, and such an animal may be obtained as a natural mutant
such as an NAR rat which does not express its own albumin.
Alternatively, the animal model may have its own gene for albumin
"knocked out" by any of the currently available genetic knock-out
techniques well known to those skilled in the art, e.g., those
techniques disclosed, for example, in U.S. Pat. Nos. 5,767,337 and
6,194,633, and in Westphal, C. H. and Leder, P.
"Transposon-Generated `Knock-Out` and `Knock-In` Gene-Targeting
Constructs for Use in Mice" Current Biology 7:R530-R533 (1997), all
of these references incorporated herein by reference. As indicated
above, in cases where the "knock out" of the animal's own gene for
albumin is to be performed in conjunction with the transfection of
the animal so as to insert the human gene for albumin, this is
preferably done by performing the "knock in" operation at the same
time so that the human albumin gene will replace the animal's
albumin gene at its proper location in the genome. As another
possibility, as also indicated above, it is possible to insert a
human serum albumin gene into the genome of the animal model
without knocking out the animal's own genetic instructions for
producing albumin, and this will result in the production of
"hybrid" mice that produce human serum albumin in addition to their
own native albumin.
[0027] In another embodiment of the present invention, an animal
model capable of more accurately reflecting the likely reaction in
the bloodstream of potential therapeutic compounds is provided
which comprises a non-human mammal which is incapable, either via a
natural mutation or via a procedure to knock out the serum albumin
gene, of producing its own serum albumin. In such animal models, it
is intended that human serum albumin may be injected into said
albumin-deficient mammal, and the resulting animal retains the
injected human serum albumin and thus may be used as an animal
model to assess drugs or other therapeutic compounds that may be
ingested by or administered to humans. In this embodiment, an
appropriate animal is selected which either has been bred not to
express serum albumin, or is a natural mutant animal, such as an
NAR rat, which does not express its own serum albumin. Still
further, it is possible to utilize an animal in which the gene for
serum albumin is knocked out via conventional genetic techniques.
In any of these cases, the animal is selected which does not
contain or express its own albumin, and the animal model in
accordance with the invention is constructed by then injecting a
suitable amount of human serum albumin in the bloodstream which is
retained in the animal's bloodstream for suitable periods to allow
the animal to be used as an animal model.
[0028] In accordance with the present invention, it is also
contemplated that animal models will be constructed which will
which reflect the various genetic diseases and disease states and
which also incorporate and express a gene for human serum albumin.
For example, an animal model in accordance with the invention may
also be constructed with any of a variety of genetic diseases,
e.g., hemophilia, sickle cell anemia, etc., or may be used to
assess pathogenic diseases, and will also express or contain human
serum albumin in its bloodstream. These animal models in accordance
with the invention will thus be particularly useful in methods of
assessing drugs utilized to treat or prevent such genetic diseases
or disease conditions caused by other pathogens.
[0029] In general, the animal models of the present invention will
thus be useful for testing of the efficacy or toxicology of drugs
or other chemicals, such as product additives, in any of the
numerous conventional methods presently used by those skilled in
the art for such testing using animal models. These tests commonly
involve the administration of a suitable amount of a drug or other
chemical agent to be assessed into an animal model such as a mouse,
rat, guinea pig or other appropriate non-human mammal followed by
the close examination of the animal model to determine the efficacy
or toxicity of the administered or injected agent. The animal
models of the present invention as described above may be utilized
in place of the models currently used and will thus be more
accurate in determining the true efficacy or toxicity of such drugs
or other chemical, physical or biological agent in humans because
the administered compound or substance will be introduced into an
environment which far more closely mimics that which will be faced
in humans.
[0030] In one particular application of the animal models of the
present invention, the models may be used to test the safety and
efficacy of albumin mutations or substitutes which may be
administered to human or animal patients to improve oxygen
transport capability or which may be used when needed, such as
during a blood transfusion in locations wherein natural blood is
scarce or unavailable. For example, the animal models of the
invention may be utilized to test blood substitutes such as the
oxygen-transporting albumin as disclosed in U.S. Pat. No.
5,948,609, incorporated herein by reference. In addition, the
models of the invention may be useful in testing other modified
forms of albumin such as the modified serum albumins disclosed in
PCT Publication WO 02/05645, also incorporated herein by
reference.
[0031] In another embodiment of the present invention, the animal
models of the invention will be useful in testing the
immunogenicity or other immunological properties of vaccines or
other materials using conventional methods in this field which
currently use animal models as a means of assessing efficacy or
reactions to new vaccines. In particular, the use of the animal
models of the present invention will be far more suitable in
assessing the immunogenic reaction in an environment such as the
human bloodstream because the animal model of the invention will
contain and/or express human serum albumin. In one particular
application of the invention, the models will be of particular use
in cases wherein molecules or compounds are coupled with albumin in
fusion proteins wherein the albumin assists in extending the
capacity of the compound to stay in the bloodstream without
dissolving. Alternatively, fusion proteins wherein a drug or other
compound is fused with human albumin are produced in which the
albumin provides a particular timed-release of the active
ingredient. In any event, the animal models of the present
invention which express human serum albumin are suitable models to
test such albumin-containing fusion proteins because they are less
likely to treat the albumin of the fusion protein as a foreign
object. Accordingly, use of the albumin models to assess the
immunogenicity or other properties or reactions of these fusion
proteins will be far more reflective of how those fusion proteins
will react in the human bloodstream. In these applications which
are suitable for the models of the present invention, it is again
contemplated that the use of the animal models of the invention
will be the same as that which is undergone for animal models which
do not express human serum albumin, and that the present invention
will thus be suitable for use in any conventional immunological or
other testing currently used in the field which employs
conventional animal models.
[0032] In summary, the animal models of the present invention can
be utilized for any of the purposes conventional animal models are
used presently, and such animals in accordance with the present
invention will afford a more accurate picture of the way that drugs
or other physical, chemical or biological agents will react in the
human body.
[0033] It is thus submitted that the foregoing embodiments are only
illustrative of the claimed invention and not limiting of the
invention in any way, and alternative embodiments that would be
obvious to one skilled in the art not specifically set forth above
also fall within the scope of the claims.
[0034] The following examples are presently only as illustrative of
certain aspects of the present invention and do not act to limit
the scope of the invention in any way.
EXAMPLES
Example 1
Injection of Albumin into NAR Rats
[0035] In accordance with the present invention, a natural mutant
rat which does not express or contain its own serum albumin, namely
an NAR rat identified as ODNAR10, was utilized as an animal model
in accordance with the present invention by injecting the animal
with suitable amounts of human serum albumin. In this example, the
NAR was given intravenous albumin injection/infusions by initially
injecting relatively small amounts of HSA solution into the animal
(1 mL). The amount of blood administered to this initial NAR rat
was monitored over a 25 day period and this monitoring showed that
a significant amount of human albumin had accumulated in the rat's
system during this time, as shown by the chromatograph of FIG. 1,
wherein column 10 represents the blood sample obtained from
ODNAR10. By the end of the treatment (when the animal expired
during anesthesia), ODNAR10 had received a total of 2.125 g HSA
administered over a 25 day period (Table I).
[0036] Following the administration of albumin to ODNAR10,
injections of HSA were given to a second NAR rat identified as
ODNAR14, this time a female who had proven barren in the breeding
program. It was estimated that if the animal's total blood volume
was 25 mL and the "normal" concentration of albumin in a mammal is
about 40 mg/mL, then she would have about 1 g of albumin in her
blood. On the assumption that the albumin in plasma represents
approximately half of the total body albumin, this rat would have
about 2 g of albumin in her total body compartments. To allow for
losses through natural turnover during the time over which albumin
would be administered (half-life of albumin=17 d), as well as some
degradation by the immune and other systems, we aimed to administer
3 g over about a 10 day period (Table II). Albumin was administered
at a rate of 2.5 ml (625 mg) every few days (see Table II) and
blood was drawn immediately prior to each injection of albumin for
SDS-PAGE analysis.
[0037] FIG. 2 shows the results of this experiment. As can be seen,
albumin is present in the serum of this rat at the first time-point
of blood collection after initiation of albumin injections (FIG. 2,
lane 9) and appears to accumulate throughout the duration of the
experiment (compare lanes 9 through 6 which represent blood drawn
after sequential albumin injections, with lane 9 showing the
results after the first bleed, lane 8 showing the results after the
second bleed and so on until the 4.sup.th bleed shown in lane 6). A
rough concentration estimate comparing the amount of albumin in the
plasma of ODNAR14 at the 4.sup.th post HSA injections bleed (FIG.
2, lane 6) with the normal amount in the plasma of a Sprague-Dawley
control rat (FIG. 2, lane 2), indicates that our procedure has
introduced approximately 25-40% of normal albumin levels into
ODNAR14's plasma. Still further albumin may be injected wherein it
is desired to increase the level of human albumin further in
accordance with the invention, and accordingly it is contemplated
that additional procedures, such as a continual infusion of albumin
over a longer time or more frequent administration or injections of
yet larger volumes, may result in still higher plasma albumin
levels. Thus, the animals with injected human serum albumin in
accordance with the present invention may suitably be used to test
or otherwise assess drugs or other compounds, such as camptothecin
compounds, and will thus be useful in replacing conventional animal
models currently used in such testing.
TABLE-US-00001 TABLE I Dates and Amounts of HSA Injected into
Pioneer NAR rat (ODNAR10) To Investigate the Distribution of HSA
into Plasma AMOUNT HSA(b) DATE(a) BLOOD DRAWN ADMINISTERED(c) 27
Feb. 2002 Pre albumin administration None 30 Apr. 2002 No 1 mL (250
mg) 02 May 2002 Yes 1 mL (250 mg) 06 May 2002 Yes 1 mL (250 mg) 10
May 2002 Yes 1 mL (250 mg) 22 May 2002 Yes 2 mL (500 mg) 25 May
2002 Yes 2.5 mL (625 mg) 28 May 2002 Died in anesthesia (a)Total
period over which albumin was administered was 25 days.
(b)Plasbumin-25; Bayer Albumin (Human) 25% (250 mg/mL), Lot number
684W027, Manufacturing date = 11 Apr. 2000, Expiry date = 11 Apr.
2003, Code = 684-71. (c)Total amount of albumin administered prior
to death of animal was 2.125 g.
TABLE-US-00002 TABLE II Dates and Amounts of HSA Injected into
Second NAR rat (ODNAR14) To Investigate the Distribution of HSA
into Plasma AMOUNT HSA(b) DATE(a) BLOOD DRAWN ADMINISTERED(c) 28
May 2002 Pre albumin administration 2.5 mL (625 mg) 30 May 2002 Yes
2.5 mL (625 mg) 03 Jun. 2002 Yes 2.5 mL (625 mg) 05 Jun. 2002 Yes
2.5 mL (625 mg) 07 Jun. 2002 Yes 2.5 mL (626 mg) (a)Total period
over which albumin was administered was 10 days. (b)Plasbumin-25;
Bayer Albumin (Human) 25% (250 mg/mL), Lot number 684W027,
Manufacturing date = 11 Apr. 2000, Expiry date = 11 Apr. 2003, Code
= 684-71. (c)Total amount of albumin administered to animal was
3.125 g.
Example 2
Transgenic NAR Rats
[0038] In accordance with the present invention, transgenic animal
models were prepared which were transfected with the gene for human
serum albumin. In this example, first generation NAR rat pups were
sent to TOSK for injection of the HSA gene within their proprietary
STEALTHGENE vector. Pups were sent to TOSK between the weights of
80 and 120 g, as per their instructions, due to their procedure
being most amenable to this age and weight of rat. From the
breeding program we have instigated for NAR rats, we have learned
that females need to be on the order of 230 g before they conceive
reliably. As of today, our heaviest females returned from TOSK have
been transfected with the gene for human serum albumin and are
185.5 g and 190 g and growing at a rate of about 15 g every 3 to 4
days. In accordance with the invention, these animals are thus
suitable as models for animal testing, and because they have been
genetically transformed, such animals will be useful in that when
bred, they will pass on the gene coding for human serum albumin to
their offspring.
[0039] In the preferred breeding program in accordance with the
invention, the usual procedures for producing and breeding a
transgenic animal will be followed. In this procedure, first the
founders will be bred to non-injected animals to produce
heterozygote animals--the F1 generation. Each of these potential
heterozygotes will be bred again to non-injected animals to produce
the F2 generation of heterozygotes. Whereas the F1 heterozygotes
will all be different from one another, each heterozygote
individual from within a single litter of the F2 generation will be
identical to its littermates with respect to the position and
integration number of the HSA gene into its genome. Heterozygous
siblings from the F2 generation will be bred together
(brother.times.sister) to produce truly homozygous animals in the
F3 generation. Other breeding strategies could be run in parallel.
For example, a compound homozygote could be produced by mating two
founders together that potentially produced more albumin in its
plasma than may eventuate from the "classical" breeding strategy.
PCR analysis can be performed on F1 and F2 individuals to establish
the presence of the HSA gene. Southern blot analysis will be used
also and especially to confirm homozygosity in the F3 generation.
In addition, conventional gel experiments can be performed to
confirm the production of human serum albumin in the transgenic
animals in accordance with the invention.
* * * * *