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DIAGNOSIS OF HYPERINSULINEMIA AND TYPE II DIABETES AND
PROTECT20N AGAINST SAME BASED ON GENES DIFFERENTIALLY
EXPRESSED IN MUSCLE CELLS (15.1)
Crass-Reference to Related. Applications
5, Anti-Aging Applications. Mice with a disrupted growth
hormone receptor/binding protein gene enjoy an. increased
lifespan. In U.S. Prov. Appl. 60/485,222, filedwJuly 8,
2003 (Kopchick8) mouse genes differentially expressed in
comparisons of gene expression in growth hormone ,
1,0 receptor/biriding.protein.gene-disrupted mouse livers and.
normal mouse livers were identified, as were corresponding
human genes and proteins. It was suggested'ahat the,human
molecules, or antagonists thereof, could be used for
protection against faster-than-normal biological aging, or .'
15 ~to achieve slower-than-normal biological aging. It was also .
taught that the human molecules may also, be used as markers
of biological aging.
In provisional application Ser. No. 60/474,606, filed
June 2, 2003 (our docket Kopchick7-USA), our research group
20 used a gene chip to.study the geneticwchanges in the liver.
of C57B1/6J mice that occur'at frequent intervals. of the,
aging process. Differential'hybridization techniques were
used to identify mouse genes that are differentially.,
expressed in~mice, depending upon their age. The level of
2f gene expression of approximately 10;000 mouse.gen~s~~(from
. the Amersham C.odelink UniSet Mouse I Bioarray, product' ~,
.code: 300013) iri. the liver of mice with average' ages of 35, ~,
~49, 56, 77, 118, 133, 207,.403, 558 and 725 days .was
determined. In essence,,complementary.RNA derived from mice
30 of different ages was screened for hybridization :~~with' ~1 ~ ~ .
oligonucleotide.probes each,specific to a particular mouse
genie, each gei~.e in turn representative of a particular mouse
gene cluster (Unigene) . 'Mouse genes which were :~ . ' , '.. . '.
differentially expressed (younger 'vs. older),~as measured.by
35 different levels ofhybridization of the respec~Cive eRNA . '
;samples with the particular probe~corresponding.to;;that.
.mouse,~gene; were identified., Related ,human genes ,.and ~ ,.
~~ ~, proteins were identified by sequence comparisons yto the'. y
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2
mouse gene or protein. In the international appl.
Kopchick7A-PCT, .filed June 2, 2004, we"added some additional
studies of CTDE-A (see below).
In a like manner, the effect of aging on the expression
of genes in mouse skeletal muscle was studied, see
provisional application Ser. No: 60/566,068, filed April 29,
2004 (our docket Kopchickl4-USA).
Anti-Diabetes Applications. In U.S. Provisional Appl.
Ser. No. 60/458,398 (our docket Kelderl-USA), filed March
31, 2003', members of our research group describe the
identification of genes differentially expressed in normal
vs. hyperinsulinemic, hyperinsulinemic vs. type II diabetic,
'or normal vs. type II diabetic mouse liver. Forward- and
reverse-substracted cDNA libraries were prepared, clones
were isolated, and differentially expressed cDNA inserts
were sequenced and compared with sequences in publicly .
available sequence databases. The corresponding mouse and
human genes and proteins were identified.
The purpose of our research group's provisional
application Ser.;No. 60/460,415 (our docket: Kopchick6-
USA), filed April 7, 2003, was similar, but complementary
RNA, derived from RNA of mouse liver, was screened against a
mouse gene chip. See also 60/506,716, filed Sept. 30, 2003
(Kopchick6.1) .
Gene,chip analyses have also been used~to identify
genes differentially expressed in normal vs.
hyperinsulinemic,~hyperinsulinemic vs. type II diabetic, or
normal~vs. type II diabetic mouse pancreas, see U.S.
Provisional Appl. 60/517,376, filed Nov. 6, 2003' ~.,
(Kopchickl2) and muscle,, see U.S Provisional Appl.
60/547512, filed Feb. 26, 2004 (Kopchickl5). .
Other differential h~rbridization applications. The use '
of differential hybridization to identify genes and proteins
is also described,in our research group's Ser. No.
. PCT/US00/12145 (Kopchick 3A-PCT), Ser. No. PCT/US00/12366 ,
(Kopchick~A-PCT); and Ser.~No. 60/400,052 ~(Kopchick5). , ;
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All of the foregoing applications are hereby
incorporated by reference in their entirety.
BACKGROUND OF THE TNVENTION
Field of the Invention
The invention relates to various nucleic acid molecules
and proteins, and their use in (1) diagnosing
hyperinsulinemia and type II diabetes, or conditions
associated with their development, and (2) protecting
mammals (including humans) against them.
Description of the Background Art
' ' '
Diabetes
A deficiency of insulin in the body results in diabetes
mellitus, which affects about 18 million individuals in the
United States. It is characterized by a high blood glucose
(sugar) level and glucose spilling into the urine due to a
deficiency of insulin. As more glucose concentrates in the
urine, more water is excreted, resulting in.extreme thirst,
rapid weight loss, drowsiness, fatigue, and possibly
dehydration. Because the cells of the diabetic cannot use
glucose for fuel, the body uses stored protein and fat for
energy, which leads to a buildup of acid (acidosis) in the
blood. If this condition is prolonged, the person can.fall
into a diabetic coma, characterized by deep'labored
breathing and fruity-odored.breath.
There are two types of diabetes mellitus, Type I and
1 Type II.'Type II diabetes is the predominant form found in
the Western world; fewer than 8% of diabetic Americans have
the type I disease.
Type I diabetes. In~Type I diabetes, formerly called
juvenile-onset or insulin-dependent diabetes mellitus, the
pancreas.cannot produce insulin. People with Type h diabetes
' must have "daily insulin'injections. But they.need to avoid
taking too'much insuliri~because'that can'lead to insulin
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shock, which begins with~a mild hunger. This is quickly
followed by sweating, shallow breathing, dizziness,
palpitations, trembling, and mental confusion. As the blood
sugar falls, the body tries to compensate by breaking down
fat and protein to make more sugar. Eventually, low blood
sugar leads to a decrease in the sugar supply to the brain,.
resulting in a loss of consciousness. Eating a sugary.food
can prevent insulin shock until appropriate medical measures
can be taken.
Type I diabetics are often characterized by their low
or absent levels of circulating endogenous insulin, i.e.,
hypoinsulinemia (1). Islet cell antibodies causing damage
to the pancreas are frequently present at diagnosis.
Injection of exogenous insulin is required~to prevent
ketosis'and sustain life.
Type II diabetes. Type II diabetes, formerly called
adult-onset or non-insulin-dependent diabetes mellitus
(NIDDM), can occur at any age. The pancreas can produce
insulin, but the cells do not respond to it.
Type II diabetes is a metabolic disorder that affects
approximately 17 million Americans. Tt is estimated that
another 10 million individuals are "prone" to becoming
diabetic. These vulnerable individuals can become resistant
to insulin,,a pancreatic hormone that signals glucose,(blood,
sugar) uptake by fat and muscle. In order to maintain normal
glucose levels; the islet cells of'the pancreas produce
more insulin, resulting in a condition called
hyperinsulinemia. .When~the pancreas can no longer produce
' enough insulin to compensate for the insulin resistance, and
thereby maintain normal glucose levels,, hyperglycemia
(elevated blood glucose) results, and~type II diabetes is
diagnosed.
Early Type II diabetics are often characterized by
~ hyperinsulinemia and resistance to insulin. Late Type'II
diabetics may be mormoinsulinemic or hypoinsulineinic. Typc
t .
II diabetics are usually not~insulin dependent or prone to
ketosis under normal circumstancesl~ ~' '
Little is known about the disease,progression from the
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normo~nsulinemic state to the hyperinsulinemic state, anal
from the hyperinsulinemic state to the Type II diabetic
state.
As stated above, type II diabetes is a metabolic
5 disorder that is characterized by insulin resistance and
impaired glucose-stimulated insulin secretion (2,3,4).
However, Type II diabetes and athe~rosclerotic disease are
viewed as consequences of having the insulin resistance
syndrome (IRS) for many years (5). The current theory of
the pathogenesis of Type II diabetes is often referred to as
the "insulin resistance/islet cell exhaustion" theory.
According to this theory, a condition causing insulin
resistance compels the pancreatic islet cells to
hypersecrete insulin in order to maintain glucose
homeostasis. However, after many years of hypersecretion,
the islet cells eventually fail and the symptoms of clinical'
diabetes are manifested. Therefore, this theory implies
that, at some point, peripheral hyperinsulinemia will~be an
antecedent of Type II diabetes. Peripheral hyperinsulinemi.a
can be viewed as the difference between what is produced by'~.
the (3 cell minus that which is taken up by the liver.
Therefore, peripheral hyperinsulinemia can be caused by
increased (3 cell production, decreased hepatic uptake or
some combination of both: It is also important to note that
it is not possible to determine the origin of insulin
resistance once it is established since the onset of
peripheral hyperinsulinemia leads,to a condition of global
insulin resistance.
Multiple environmental and genetic factors are involved
in the development ofinsulin resistance,,hyperinsulinemia
and type II diabetes. An important risk factor for the
development of insulin resistance, hyperinsulinemia and type
II diabetes is obesity, particularly visceral' obesity
' (6,7,8).~Type IT diabetes exists world-wide, but in
developed societies, the prevalence has risen as the average
age of the population increases and the average individual
becomes more obese. . ' ,
,, .
Obesity and Diabetes. Obesity is a'aerious and growing~~
. . , ~ ',
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problem in the United States,. Obesity-related health risks
include high blood pressure, hardening of the arteries,
cardiovascular disease, and Type TI diabetes (also known as
non-insulin-dependent diabetes mellitus, Type II
diabetes)(9,10,11). Recent studies show that 850 of the
individuals with Type IL diabetes are obese (12).
Treatment of Diabetes. For many years, treatment was
insulin therapy for Type I and oral sulfonylureas and/or v
insulin therapy for Type II. Metformin (glucophage) was the
first antidiabetic drug approved by FDA (May 1995) for the ,
treatment of Type II diabetes since the oral sulfonylureas
were introduced in 1984. Metformin promotes the use of
insulin already in the blood. This May 1995 approval was
followed by the September 1995 approval of another'
antidiabetic drug, Acarbose (precose). It slows down the
digestion and absorption of complex sugars, which reduces
blood sugar levels after meals. '
Before 1982, insulin was purified from beef or pork
pancreas. This was a problem for those diabetics allergic to
animal insulin. Researchers produced a synthetic insulin
called humulin. Approved by FDA in 1982, it was the first
genetically engineered consumer health product manufactured
for diabetics. Synthetic insulins can be produced in
unlimited quantities. . ~~,
Another possible treatment for diabetes includes
surgically replacing the pancreas' endocrine tissues,(islets
of Langerhans) with healthy islet of Langerhans tissue
grafts. Since 1988, 45 patients worldwide have undergone ,
30'~ successful transplantation. ' y ,
.;
fompliaations. Complications of diabetes (end~.organ
damage) include.retinopathy, neuropathy, and neph~opathy
(traditionally designated as microvascular complications) as
~ well as atherosclerosis (a macrovascular complication). ;. '
Early stages~of hyperglycemia~can usually be controlled~by .
an alteration~in~diet and increasing the amount of exercise,
.. .
but drug treatment, including insulin, may be required: It
haslbeen shown that meticulous blood glucose control~can
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often slow down or halt the progression of diabetic
complications if caught early enough (1). However, tight
metabolic control is extremely difficult to achieve.
Animal Models
Transgenic Mouse Models of Dia3aetes or Diabetes
Resistance. McGrane, et al., J. Biol. Chem. 263:11443-51
(1988) and Chen, et al., J.,Biol. Chem.~, 269:15892-7 (1994)
describe the genetic engineering of mice to express bovine
growth hormone (bGH) or human growth hormone (hGH),
respectively. These mice exhibited an enhanced growth
phenotype. They also developed kidney lesions similar to
those seen in diabetic glomerulosclerosis, see Yang, et al.,
Lab. Invest., 68:62-70 (1993). Ogueta, et al., J.
Endocrinol., 165: 321-8 (2000) reported that transgenic mice
expressing bovine GH develop arthritic disorder and self-
antibodies.
Growth hormone has many roles, ranging from regulation
r 20 of protein, fat and carbohydrate metabolism to growth
promotion. GH is produced in the somatrophic cells of the
anterior pituitary and exerts its effects either through the
GH-induced action of IGF~-I, in the case of growth promotion,
or by direct interaction with the GHR on target cells
including liver, muscle, adipose, and kidney cells.
Hypo'secretion of GH during development leads. to dwarfism,
and hypersecretion before puberty leads to gigantism. In
adults, hypersecretion of ,GH results in acromegaly, a
clinical condition characterised by enlarged facial bones,
hands, feet, fatigue and an increase in weight. Of those
individuals with acromegaly~ 25% develop type II diabetes.
This may be due to.insulin resistancecaused by the high
circulating levels of GH leading to high circulating levels
of insulin (Kopchick~et,al., Annual Rev. Nutrition 1999.
19:437-61) .' ~ '
A further mode of GH.action may be~through the
,, transcriptional regulation of a number of genes contributing
. to the' physiological effects of GH. .
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Growth hormone genes and the proteins encoded by them
can be converted into growth hormone antagonists by
mutation, see Kopchick USP 5,350,836. Transgenic mice have
been made that express the GH antagonists bGH-G119R or hGH
G120R, and which exhibit a dwarf phenotype. Chen, et al.,
J. Biol. Chem., 263:15892-7 (1994); Chen, et al., Mol.
Endocrinol, 5:1845-52 (1991); Chen, et al., Proc. Nat. Acad.
Sci..USA 87:5061-5 (1990). .These mice did not develop
kidney lesions.. See Yang (1993), supra. '
Chen, et al., Endocrinol, 13&:660-7 (1995) compared the
effect of, streptozotocin treatment in normal nontransgenic
mice, and in mice transgenic for (1) a GH receptor
antagonist, the G119R mutant of bovine growth hormone or (2),
the E117L-mutant of bGH. (According to Chen's ref. 24,~.
these large GH transgenic streptozotocin-treated mice
constitute an animal model for diabetes.) Glomerulosclerosis
was seen in diabetic (STZ-treated) nontransgenic mice and in
diabetic bGH-E1.17L mice, but not in diabetic bGH-G119R (GH
antagonist) mice.
Two of the proteins which mediate growth hormone
activity are the growth hormone receptor and the growth
hormone binding protein, encoded by the same gene in
mice(GHR/BP). It is possible to genetically engineer mice
so that the gene encoding these,proteins is disrupted
' ("knocked-out"; inactivated), see Zhou, et al.,,Proc. Nat.
Acad. Sci. (USA), 94:13215-20 (1997). Zhou, et al.
inactivated the GHR/BP gene by replacing the 3' portion of
. ~exon 4 (which encodes a portion of the GH,binding domains)
and the 5'~region of intron 4 with a~neomycin gene cassette.
The modified gene,was introduced into the target mice by
homologous recombination. Like mice expressing a'GH
antagonist,. homozygous GHR/BP-KO mice exhibit a dwarf
phenotype., GHR/BP-KO mice, made diabetic by streptozotocin
' treatment, are protected from the development of diabetes-
associated nephropathy. Bellush,~et al.,'Endocrinol.,
' 141:163-8 (2000) . '
..
High-Fat Diets. High-fat diet.s~have been shown to
induce~both. obesity~,and Type II diabetes'in laboratory
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9
animals (13). Surwit and colleagues demonstrated that male
C57BL/6J mice are extremely sensitive to the diabetogenic
effects of a high-fat diet when initiated at weaning. At
six months.of age, high-fat fed animals had significantly
elevated fasting blood-glucose and insulin levels and also
demonstrated a decrease in insulin sensitivity (14). Ahren
and colleagues (15) reported evidence of insulin resistance
as well as diminished glucose-stimulated insulin release,
after feeding with a high-fat diet for 12 weeks. These mice
also showed elevated levels of total cholesterol,
triglycerides, and free fatty acids, another hallmark of
Type II diabetes.
Anatomy aad Physiology of Muscle
Muscle tissue constitutes about 40% of the body mass.
Muscles may be classified by location, i.e., skeletal if
attached to bone, cardiac if forming the wall of the heart,
and visceral if associated with another body organ. Muscles
may also be classified as voluntary or involuntary,
depending on how~their contractions and relaxations are
controlled. Skeletal muscles are voluntary, while cardiac
and visceral muscles are involuntary. It is also possible
to classify muscles morphologically; skeletal and cardiac
25, muscle cells are striated, whereas visceral muscle cells are
not. .
Each skeletal. muscle i's composed of many individual muscle
cells called muscle fibers. The fibers are held together by
fibrous connective-tissue membranes called fascia. The'.
fascium which'envelops the entire muscle is'the.epimysium
and the fascia which penetrate the muscle,,separating the
fibers into bundles ~(fasciculi),are called perimysium. ,Very
thin fascia (endomysium) sheath each.muscle fiber. Skeletal
muscles are attached either directly to a bone, or
indirectly through a~tendon.~
The individual'mu~scle fibers'(cells) comprise threadlike
protein 'structures called myofibrils.
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There are over,600 muscles in the human body. We will have
occasion later to refer to the gastrocnemius. Tt is a
superficial muscle in the posterior compartment of the lower
leg, which together with the underlying soleus forms the
5 characteristic bulge of the calf.
Role of Muscle is Development of Type II Diabetes
Muscle, fat and liver tissues are the major
contributors to the development of insulin resistance,
10 hyperinsulinemia, and, ultimately, type II diabetes.
Muscle cells respond to insulin by increasing glucose uptake.
from the bloodstream. Muscle tissue can become resistant to
insulin, causing the beta cells to initially increase
insulin secretion. Eventually, though, the beta cells become
unable to compensate for this increasing insulin resistance
from muscle and other cells, and they fail to respond to
elevated blood glucose levels. Thus, clinical type 2-
diabetes results from the combination of insulin resistance
and impaired beta cell function.
Defects in muscle glycogen synthesis are known to play
a role in the development of insulin resistance. At least
three steps-those mediated by glycogen synthase, hexokinase,
and GLUT4-have been reported to be defective in patients
with type 2 diabetes. ~ '
Fatty acids can induce insulin resistance, and it has
been suggested that this was a consequence of altered
insulin signaling through PI3-kinase. PKC-theata has. also
been implicated. '
See generally Pet.ersen, et al.,, "Pathogenesis of
Skeletal muscle insulin resistance in type 2 diabetes
',
mellitus", in "A Symposium: Evolution of type 2 diabetes
mellitus management", at Amer. J. Cardiol., 90(5A): 11G-18G~,
(Sept. 5, 2002).
Adverse Effects of Type IT Diabetes on Muscle
"Myopathy is a general term used to describe any ~~~ , ,
',
disease of muscles, such as the, muscular dystrophies;and
myopathies associated.with thyroid disease. It.can be caused ~. ',
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11
by endocrine disorders, including diabetes, metabolic
disorders, .infection or inflammation of the muscle, certain
drugs and mutations in genes. In diabetes; myopathy is
thought.to be caused by neuropathy, a complication of
diabetes. General symptoms of myopathies include muscle
weakness~of limbs sometimes occurring during exercise
although in some cases the symptoms diminish as exercise
increases. Depending on the type of myopathy, one muscle
group may be more affected than others." See "Joint and
Mug cle Problems Associated with Diabetes",
www.iddtinternational.orcr/-iointandmuscleprbblems html [Last
modified June 12, 2003] .
Diabetic muscle.,infarction can spontaneously affect '
pat Tents with a long history of poorly controlled diabetes.
"Most affected patients have multiple microvascular
complications (neuropathy, nephropathy, and retinopathy).
Tha clinical presentation is an acute onset of pain and
swat ling over days to weeks in the affected muscle groups
(usually the thigh or calf), along with varying degrees- of
tenderness....~Therapy consists of rest and analgesia.
Routine daily activities are not deleterious to the
condition, but physical therapy may cause exacerbation.
Spontaneous diabetic muscle infarction tends to resolve over
25, ~a period of weeks to months in most cases." See
"Musculoskeletal Complications of Diabetes - Part 2",'
ww~nr.diabetic=lifestyle.com/articles/jan02 whats-1 htm [last
modified Feb., 9,~ '2004] . See also Trujillo-Santos, et al.,
"Diabetes muscle infarction: an underdiagnosed complication
.of long-standing diabetes," Diabetes Care, 26(1):211-5
(2p03) . , , .
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Ider~,tificatioa of genes involved in, hyperinsulir~.emia and
type II diabetes, generally
Our attention recently has focused on the generation of
muscle mRNA expression profiles and the identification of
genes involved in the genesis of, the obesity-induced
hyperinsulinemia and type-II diabetes. To date, no one has
attempted to study the actual progression from the. normal
condition to that of hyperinsulinemia or from
hyperinsulinemia to Type II diabetes in an attempt to
identify genes that are up-regulated or down-regulated in
muscle as the disease progresses. ,
In previous studies aimed at identifying genes involved
i n diabetes-induced glomerulosclerosis, differential display
and traditional subtractive hybridization techniques were
used (16-20). While effective for the identification of a
few genes (e. g. hmuncl3, PED/PEA-15, lactate dehydrogenase,
amiloride sensitive sodium channel, ubiquitin-like protein,
mdr 1, and a-amyloid protein precursor as well as a few
novel genes) these techniques can be quite labor intensive.
The PCR-based method of subtractive hybridization requires
1 ess starting material, and allows the simultaneous
isolation of all differentially expressed cDNAs into two
groups (up-regulated and down-regulated) . ,
However,' the PCR=based method of subtractive
hybridization is also quite labor-intensive,, produced large
numbers of false positive candidates and ultimately resulted
in the identification of a relatively limited number of
differentially expressed genes. (see Kelderl-USA
application) .
~, In order to expand the number of genes that can be
analyzed, simultaneously,'several groups have begun to
utilize DNA microarray analysis to measure differences in
gene expression between normal and diseased'states.
However, these experiments.have been limited in regards to
the number of,experimental~conditions analyzed. DNA
mi croarray.anafysis has been performed on.normal, obese and
ds.abetic mice (21)~'v Also, the obesity and diabetes in the
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13
mouse models examii~.ed were caused by a specific endogenous
genetic mutation (22). The differentially expressed genes
in the above models may be very different from genes
differentially expressed due to diet-induced obesity and
Type-II diabetes.
The use of differential expression and related techniques to
identify genes useful in the treatment of diabetes has been
rwiyewed.by Perfetti, et al., Diabetes Technol. &
Therapeut . , 5 ( 3 ) : 421-3 . ( 2 0 03 ) . Bernal -Mi zrachi , . et al . ,
D~.abetes Metab. Res. Rev. 19: 32-42 (2003) . .
Other papers of interest include:
Wada, et al., "Gene expression'profile in'
streptozotocin-induced diabetic mice kidneys undergoing
glomerulosclerosis", Kidney Int, 59:1363-73 (2001);
Song, et al., "Cloning of a novel gene in the human
ks.dney homologous to rat muncl3S: its potential role in
diabetic nephropathy", Kidney Int:, 53:1689-95 (1998);
' Page, et al., "Isolation of. diabetes-associated kidney
genes using differential display", Biochem. Biophys. Re's.
Comm. , 232 :49-53 (1997) .
Peradi,,"Subtractive hybridization claims: An efficient
technique to detect overexpressed mRNAs in~,diabetic
nephropathy," Kidney Int. 53:926-31 (1998).
'Condorelli, EMBO J., 17:3858-66 (1998).
., '
Diabetes-Specific Differential Expression in Muscle
Sreekumar, et al., "Gene expression profile in skeletal
msucle of type 2 diabetes and the effect of insulin
treatment," Diabetes 51:,: 1913 (June 2002) surveyed 6,451
geneses,. and identified 85 genes for which there was an
alteration in skeletal muscle transcription in diabetic
~35 patients after withdrawal of insulin treatment. Subsequent
insulin~treatment resulted in further' changes in
transcription of 74 of ,the 85 , genes (15 increased, 59: '
da creased), and'also resulted~.in alteration of 29'additional
gene.,transcripts. '
.: ,.
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14
Mootha, et al., "PCG-lcx responsive genes involved in
oxidative phosphorylation are coordinatively downregulated
in human diabetes," Nature Genetics 34 (3) ; 267 (July 2003) ,
used DNA microarrays to detect changes in the expression of
sets of related genes, rather than of individual genes. They
classified over 22,000 genes into 149 data sets; some of
these data sets overlapped. They looked for a statistical
correlat ion between the overall~rank order of the genes in
differential expression, and the groups to which the genes
belonged. Expression was compared pairwise among three
groups: males with normal glucose tolerance; males with
impaired glucose tolerance; and males with type 2 diabetes.
The set with the highest enrichment score (the one whose
members ranked highly most often relative to chance
expectat ion) was an internally curated set of 106 genes
involved in oxidative phosphorylation. While the average
decrease for the individual genes was modest (~20%), it was
also consistent, being observed in 89% (94/106) of the genes
in quest ion. This paper is reviewed by Toye and Gauguier,
"Genetics and functional ~genomics of type -2 diabetes
mellitus", Genome Biology, 4: 241 (2003) .
Patti, et al., "Coordinated reduction of genes of oxidative
metabolism in humans with insulin resistance and diabetes:
Potential role of PGC1 and NRFl~~, Proc. Nat. Acad: SCi.
(USA) , 100 (14) : 8466 (July ~ 8, 2003) used microarrays to
analyze skeletal muscle exprelssion of'genes in nondiabetic
insulin- resistant subjects at high risk.for diabetes (based
on family hisotry of diabetes end Mexican-American
ethnicity) ,and diabetic Mexican-American subjects., Of 7,129
sequence s represented on;the microarray, 187..were
differentially expressed between'control, and diabetic
subjects. However, no single gene remained significantly
differentially expressed after controlling fog multiple
comparis on false discovery by' using the Benjamini-Hochberg '
' methods see.Benjamini, et'al., J. R. Stat. S,oc. Sert. B.
' .
57:289-300 (1995); Dudait,~'et al., 'Stat. Sin. 12: 111-139
(2002). Consequently, Patti et al.,,sought to identify.
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groups of related genes,with similar patterns of
differential expression using MAPP FINDER and ONTOEXPRESS.
According to MAPP FINDER, the top-ranked cellular component
terms were mitochondrion, mitochondrial~membrane,
5 mitochondrial inner membrane, and ribosome, and the top-
ranked process term was ATP biosynthesis. According to
ONTOEXPRESS, the over-represented groups were energy
gene ration, protein biosynthesis/ribosomal proteins, RNA
binding, ribosomal structural protein, and ATP synthase
10 complex.
Huang, Xudong, "Identification of abnormally expressed genes
in skeletal muscle contributing to insulin resistance and
type 2 diabetes", Thesis, document id: 9576 Lunds
15 University 2002, reported differential expression of the
mitochondrially-encoded ND1 gene iwhuman diabetic patients
and of the nuclear-encoded cathepsin L gene in mice.
Standaert, et al., ":Skeletal muscle insulin.resi.stance in
obes~.ty-associated type 2 diabetes in monkeys is linked to a
defect in insulin activation of protein kinase ~C-
zeta/lambda/iota Diabetes 51: 2936 (Oct. 2002). the authors
concluded that defective activation of atypical PKCs played
an important role in the patehogenesis of peripheral.insulin~'
resistance in both obese prediabetic and diabetic monkeys. '.
'They attributed this linkage to the apparent requirement for
aPKCs during insulin-stimulated glucose transport.
lSrommer, et al., Arn. J. Physiol.,"Skeletal muscle insulin
resin tance after trauma: insulin signaling and glucose
transport°', 275 (2 Pt. 1) : E3518 (Aug. 1998) concludedl'th.at
insulin resistance in skeletal muscle after surgical trauma
is associated with reduced glucose transport but. not with
impaired glucose signaling to PI 3-kinase om its downstream
~.. 35 target, .Akt. ;, .
Agixig-Specific Differential Expression in Muscle
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16
Gene Chip-Based Identification of genes involved in aging of
skeletal muscle
Several groups have used DNA microarrays to measure
differences in gene expression. caused by the aging process.
However, then a experiments are extremely limited in regards
to the number of aging time points or experimental
conditions.
Weindruch, et al . , "Microarray profiling of gene
expression ire aging and its alteration by caloric
restriction i.n mice" in Symposium: Calorie Restriction:
effects on Body Composition, Insulin Signaling and Aging
9185-9235 (20 01)(21) compared expression in gastrocnemius
muscle from 5- and 30-month old C57BT,/6 mice, with and
withoutlcaloric restriction. In this analysis, the
expression of 113 genes was found to be changed by at least
two-fold in 5-month old mice compared to 30-month old mice.
Caloric restriction of comparable mice caused a reversal of
the altered gene expression of 33 genes.
Of the 6 347 genes surveyed in the oligoilucleotide
. microarray, only 58 (0.90) displayed a greater than 2 fold
increase in gene expression as~a function of aging, whereas
55(0.9%) displayed a greater than 2 fold decrease.
Of the genes positively correlated with aging, 160
could, be assigned to stress responses. The largest
'25 differential expression between young and aged animals (3.8
fold) was the mitochondrial sarcomeric creative kinase. '
~Of the genes negatively correlated with. aging, 13% were '
involved in energy metabolism. A noteworthy number were
genes encoding biosynthetic enzymes (cytochrome P450 IIC12,
squaelene.synthase, stearoyl-CoA desaturase, EF-1-gamma. ~,,
Another down regulator was a CpG binding protein, MeCP2.
''Weindruch further reported that age-related changes in
gene expressi.on.profile were "remarkably attenuated" by
caloric restriction. ~ , ,
35~ , What'app ears to be the same experiment is, discussed in
Lee,,'et al., "Gene expression profile of aging and, its
retardation by caloric restriction," Science, 285: 1390 (Aug. ;;
27,, 1999)': ,This papers lists the individual genes which.
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17
were different Tally expressed by more than 2-fold, and
classifies them as energy metabolism, neuronal factors,
protein metabolism, stress response, biosynthesis, calcium
metabolism or DNA repair genes.
Welle, et al., "Skeletal muscle gene expression profiles
in 20-29 year old and 65-71 year old'women," Exper. .
Gerontol., 39: 369-77 (2004) and available electronically as
doi:10.1016/j.exger.2003.11.011 studied gene expression and
physical cond~.tion in seven young and eight older women.
With respect to physical condition, the measured or
calculated parameters were total body mass, lean body mass,
left leg lean mass (by biopsy), maximum isometric left knee
extension force, left knee extension force/left keg lean
mass, Peak V02/lean body mass, and Peak VOZ/left leg lean
mass.
There were 1178 "probe sets" (representing 1053
different Unigene clusters) for which differential
expression was detected; 550 for which expression was higher
in older women, and 628 the inverse effect. The differences
ranged from 1 _ 2 to 4 fold; most (78Ao) were less than 1.5
fold. The complete list of-differentially expressed genes is
given in the Rochester Muscle database website,
www.urmc.roche ster.edu/smd/crc/swindex (".html" omitted, in
accordance with USPTO requirements, so that the publication
v25 of this application will not create~an active hyperlink).
The gene most highly overexpressed in older muscle was
p21 (cyclin-dependent kinase inhibitor 1A) (4.01 fold)'. This
one~of several genes (see Welle Table 2) which are
potentially related to, DNA damage and repair. Welle also
thought it not eworthy how many of the differentially
expressed genes were, ones that encode proteins which bind to
pre-mRNAs~-or mRNAs (see Welle Table 3).
35, ~ Other DifferentialeSubtraetive Hybridization Studies of
Interest ~ . ,
. ,. ~ . . . '
hang, et al., Kidney International, 56:5'49-558 (1999)
identified genes up-regulated in 5/6:nephrectomized
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18
(subtotal renal ablation).. mouse kidney by a PCR-based
subtraction method. Ten known and nine novel genes were
identified. The ultimate goal was to identify genes',
involved in glomerular hyperfiltration and
hypertrophy.Melia, et al., Endocrinol., 139:688-95 (1998)
applied subtractive hybridization methods for the
identificati on of androgen-regulated genes in~mouse kidney.v
The treatment mice were dosed with dihydrotestosterone, an
androgen. Kidney androgen-regulated protein gene was used
as a positive control, as it is known to be up-regulated by
DHT.
See als o Holland, et al., Abstract 607, "Identification
of Genes Possibly Involved in Nephropathy of Bovine Growth
Hormone Transgenic Mice" (Endocrine Society Meeting, June 22,
2000) and Coschigano, et al., Abstract 333, "Identification
of Genes Pot entially Involved in Kidney Protection During
Diabetes"'(Endocrine Society Meeting, June 22; 2000)'.
The following differential hybridization articles may
also be of~interest: Wada, et al., "Gene expression profile
in streptozotocin-induced diabetic mice kidneys undergoing
glomerulosclerosis", Kidney Int, 59:1363-73 (2001); Song, et
al., "Cloning of a novel gene in the human kidney homologous
to rat muncl3 S: its potential role imdiabetic nephropathy",
Kidney Int., 53:1689-95 (1998); Page, et al., "Isolation
of diabetes-associated kidney genes using differential
display", Biochem. Biophys. Res. Comm., 232:49-53 (1997);
Peradi, "Subtractive hybridi,zation'claims: An efficient '
technique to detect overexpressed mRNAs in diabetic
nephropathy," Kidney Int~..53:926-31 (1998); Condorelli,
EMBO J., 17:3858-66,(1998). ~ .'
Apoptosis and CIDE-A . . '
Apoptosis is arform of programmed cell death that
occurs in an active and controlled manner to eliminate v '
unwanted cells.,- ;Apoptotic cells undergo an o~rch.estrated,,
~,cascade.of morphological changes such as. membrane bhebbing,
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19
nuclear shrinkage, chromatin condensation, and formation df
apoptotic bodies which then undergo phagocytosis by
neighboring cells _ One of the hallmarks of cellular
apoptosis is the cleavage of chromosomal DNA into discrete
oligonucleosomal size fragments. This orderly removal of
unwanted cells minimizes the release of cellular components
that may affect neighboring tissue. In contrast, membrane
rupture and release of cellular components during necrosis
often leads to tis sue inflammation.
The process of apoptosis is highly conserved and
involves the activation of the caspase cascade. Cohen, GM.
(1997) Caspases: the executioners of apoptosis. Biochem.
J. 326:1-16; Budihardjo, I., Oliver, H., Lutter, M., Luo,
X., Wang, X. (19 9 9) Biochemical pathways of caspase
activation during apoptosis. Annnu. Rev. Cell. Dev.
Biol.15:269-290; ~'acobson, M.D., Weil, M., Raff, M.C.
(1997) Programmed cell death in animal development. Cell
88:347-354. Caspases are a family of serine proteases that
are synthesized as inactive proenzymes. Their activation by
20-. apoptotic signals such as CD95 (Fas) death receptor
activation. or tumor necrosis factor results in the cleavage
of specific target proteins and execution of the apoptotic
program. Apoptosi_s may occur by either an extrinsic pathway
involving the acti_vatzon of cell surface death receptors
(DR) or by an intrinsic mitochondrial pathway. Yoon, J-H.
Gores G.J. (2002) Death receptor-mediated apoptosis and
the liver. J. Hep atology 37:400-410.
These pathways are not~mutually exclusive and some
cell types require the activation of both pathways for
maximal apoptotiC signaling. In type-I cells, death
v.
receptor'activation,leads to the recruitment and activation
of caspases-8/10 and the rapid cleavage and activation; of.
caspase-3 in a mit ochondrial-independent manner.w .~
Hepatocytes are members of the Type-II cells in which
mitochondria are a ssenti~al for DR-mediated., apoptosis ~ .
Scaffidi, C~. , Fulda, 5. , Srinivasan, A.. ,, Friesen; C. , ,Li,
F., Tomaselli, K.J ., Debatin, K.M.; Krammer, P.H.,~Peter,
,, ' , ,,
M.E. (1998) Two :CD95 (APO-1/Fas) ~signalin~ pathways. EMBO
J. 17:1675-1687, Zn this,,pathway,~ahe:pro-apoptotic protein
. , , ' '~ , , .
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Bid is truncated by activated caspases-8/10 and translocates
to the.mitochondria. Luo, X., Budihardjo, I., Zou, H.,
Slaughte r, C., Wang, X. (1998) Bid, a Bcl2 interacting
protein, mediates cytochrome c release from mitochondria in
5 response to activation of cell surface death receptors.
Cell 94:481-490; Li, H., Zhu, H., Xu, C.J., Yuan, J.
(1998) Cleavage of BID by caspase 8 mediates the
mitochondrial damage in the Fas pathway of apoptosis. Cell
94:491-5 O 1. This translocation leads to mitochondrial
10 cytochrorne c release and eventual activation of caspases-3
arid 7 vi a cleavage by activated caspase-9.
One of the substrates for activated caspase-3 is
the DNA fragmentation factor (DFF). DFF is composed of a 45
kDa regulatory subunit (DFF45) and a 40 kDA catalytic
15 ' subunit (DFF40). Liu, X., Zou, H., Slaughter, C., Wang,
X. (199'0 DFF, a heterodimeric protein that functions
downstream of caspase-3 to trigger DNA fragmentation during
apoptosis. Cell 89:175-184.~DFF45 cleavage by activated
caspase-3 results in its dissociation from DFF40 and allows
20 the caspase-activated DNAse (CAD) activity of DFF40 to
'cleave ch.romosomal,DNA into ~ligonucleosomal size fragments.
Liu, X.1, Li, P., Widlak, P., Zou, H., Luo, X., Garrard,
W.T., Wang, X. (1998) The 40-kDa subunit of DNA
fragmentation factor induces DNA fragmentation and chromatin
condensat ion during apoptosis. Proc. Natl. Acad. Sci. USA.
95:8461-8 466; Halenbeck, R., MacDonald, H., Roulston, A.,
Chen, T.T., Conroy, L., Williams, L.T. (1998) CPAN, a human
nuclease, regulated by the caspase-sensitive inhibitor
,. DFF45. Curr Biol. 8:537-540; Nagata, S. (2000) Apoptotic
DNA fragmentation. Exp. Cell Res. 256:12-8.
Recently, a novel family of cell-death-inducingw ~~
DFF45-like effectors'(CIDEs) have been identified that. .
includes CIDE-A, CTDE-B and CIDE-3/FSP2. Inohara, N., ,
Koseki, T., Chen, S., Wu, X., Nunez, G. (1998) CIDE, a
'35 novel family of cell death activators with homology to the
45 kDa aubunit of the DNA fragmentation factor.' EMBO J.
,.17:2526-2 533; Danesch, U., Hoeck, W., Ringold; G.M. (1992)
Cloning and transcriptional regulation of a novel adipocyte- -
specific gene, FSP27. CAAT-enhancer-binding protein (C/EBP)
'~
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21
and C/EBP-life proteins interact with sequences required for
differentiate on-dependent expression. J. Biol. Chem.
267:7185-7193 ; Liang, L., Zhao, M., Xu, Z., Yokoyama, K.K.,
Li, T. (2003) Molecular cloning and characterization of
CIDE-3, a novel member of the cell-death-inducing DNA-
fragmentation.-factor (DFF45)-like effector family. Biochem.
J. 370:195-203.
The CIDE s contain an N-terminal domain that shares
homology with the N-terminal region of DFF45 and may
represent a regulatory region via protein interaction. See
Inohara., supra; Lugovskoy, A.A., Zhou, P:, Chou, J.J.,
McCarty, J.S., Li, P., Wagner, G. . (1999) Solution
structure of the CIDE-N domain of CIDE-B and a model for
CIDE-N/CIDE-N interactions in the DNA,,fragmentation pathway
of apoptosis. Cell 9:747-755. The family members also
share a C-terminal domain that is necessary and sufficient
for inducing cell death and DNA fragmentation; see Inohara
supra. The overexpression of CIDE-A induces cell death that
can be inhibited by DFF45. However, CIDE-A-induced
apoptosis,is not inhibited by caspase-8 inhibitors thereby
suggesting the presence of additional, caspase-independent,
pathway (s) for 'the induction of apoptosis, see Inohara
supra. Previous reports have indicated that human and mouse
CIDE-A are expressed in several tissues such as brown
adipose tissue (BAT) and heart and are localized to the
mitochondria, Zhou, Z., Yon Toh, S., Chen, Z., Guo, K., Ng,
C.P., Ponniah, S., Lin, S.C., Hong, W., Li, P. (2003)
Cidea-deficient mice have lean phenotype and are resistant
to obesity. Nat. Genet. 35:49-56. . In addition to the
ability to induce apoptosis, CIDE-A can interact and inhibit.
'~UCP1 in BAT and may therefore play a role in regulating
energy ibalance, see Zhou supra.
Previous reports have indicated that CIDE-A is not
expressed.in either adult human or mouse liver tissue, see
Inohara supra, Zhou supra.
,, ,The human protein cell death activator'CIDE-A is of
particular'int erect because of its highly dramatic Change em
liver expressson with age,.first demonstrated in our '
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22
Kopchick7 app Iication, supra. CIDE-A expression is elevated
in older normal mice. CIDE-A expression was studied for
normal C57BI/ 6J mouse ages 35, 49, 77, 133, 207, 403 and 558
days. Expression is low at the first five data points,
then rises sharply at 403 days, and again at 558 days.
CIDE-A was therefore classified as an "unfavorable
protein", i:e_, it was taught that an antagonist to CIDE-A
could retard biological aging.
In Kopchick7A-PCT we reported that CIDE-A is also
prematurely expressed in hyperinsulinemic and type-II
diabetic mouse liver tissue. CIDE-A expression also
correlates with liver steatosis in diet-induced obesity;
hyperinsulinernia and type-II diabetes. These observations
suggest an additional pathway of apoptotic cell death in
~ Non-Alcoholic Fatty Liver,Disease (NAFLD) 'and that CIDE-A
may play a rot a in this serious disease and potentially in
liver dysfunct ion associated with. type-II diabetes.
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SUMMARY OF THE INVEIJ'fION
Differential hybridization techniques have been used to
identify mouse genes that are differentially expressed in
the muscle (gastrocnemius) of mice, depending upon their
development of hyperinsulinemia or type II diabetes.
In essence, complementary RNA derived from normal mice,
or mouse models of hyperinsulinemia or type II diabetes, was
screened for hybridi nation with oligonucleotide probes each
specific to a particular mouse database DNA, the latter
being identified, by database accession number, by the gene
manufacturer. Each database DNA in,turn was also identified '
by the gene chip manufacturer as representative of.a
particular mouse gene cluster ,(Unigene).
In most cases,. this database DNA sequence is a full
length genomic DNA or cDNA sequence, and is therefore either
identical to, or otherwise encodes the same protein as does,
a natural full-length genomic DNA protein coding sequence.
Those which don't pre sent at least a partial sequence of a
natural gene or its cDNA equivalent.
For the sake of simplicity, all of these mouse database
DNA sequences, whether full-length or partial, and whether
cDNA or genomic DNA, are referred~to herein as "mouse genes".
When only the genomic sequence is intended, we will refer
specifically to "genomic DNA" 'or "gDNA" .
The sequences in the protein databases are determined w
either by directly sequencing the protein or, more commonly,
by sequencing a DNA, and,then determining the translated
amino acid sequence in accordance~with the Genetic Code. All
of the mouse sequences in the mouse polypeptide database are
referred to herein as "mouse proteins" regardless of whether',
they are in fact full length, sequences.
' Mouse genes whi~ch~were differentially expressed (normah
vs. hyperinsulinemic,,hyperinsulinemic'vs. diabetic, or
normal vs. diabetic), as measured by.different levels of
, hybridization of the respec ive cRNA samples with the
particular. probe corresponding to that mouse gene) were
identified.
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Since the progre scion is from normal to
hyperinsulinemic, and thence from hyperinsulinemic to type
II diabetic, one may define mammalian subjects as being more
favored or less favored, with normal subjects being more
favored than hyperinsulinemic subjects, and hyperinsulinemic
subjects being more favored than type II diabetic subjects.
The subjects' state may then be correlated with their gene
expression activity.
The terms "normal" and "control" are used' ,
interchangeably in this specification, unless expressly
stated otherwise. The control or normal subject is a mouse
,which is normal vis-a-vis fasting insulin and fasting
glucose levels. The term "normal", as used herein, means
normal relative to those parameters, and does not
necessitate that the mouse be normal in every respect.
A mouse gene is said to have exhibited a favorable
behavior if, for a particular mouse age of observation, its
average level of expression in mice which are in a more ,
favored state is higher than that in mice which are in a
less favored state. A mouse gene is said to have exhibited
an unfavorable behavior if, for a particular mouse age of
observation, its average level of expression in mice which
are in a more favored state is lower than that in mice which
are in a less favored state. ,
When we observe the mice at several different ages, it.
is possible for their expression behavior to. vary from time
point to time point. For a given comparison. of subjects,
e.g.,~ normal vs. hyperinsulinemic, we classify.the mouse .
gene as favorable or unfavorable on the basis of the;
direction of the largest expression change, and it is the
magnitude of this largest~expression change,~expressed as a w
ratio of greater to 1 esser, which is set forth in the'Master ,
Table 1 data for that mouse gene. Thus, if,at 2 weeks,~there
was a 3-fold favorabl a behavior, and at 8 weeks, there was a
4-fold unfavorable behavior, and~at all other observed, time
points, the behavior was weaker than 3-fold, the mouse~gene
would be~classified as an unfavorable gene,with respect.to ~,'
'the subject comparison in question.
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It will be appreciated that it may be that if the mouse
gene were observed at an age other than one of the ages
noted in the Exampleso we would have observed a still
stronger differential expression behavior. Nonetheless, we
5 must classify the mouse genes on the basis of the behavior
which we actually observed, not the behavior which. might
have been observed at' some other age.
We are particularly interested in mouse genes which
10 exhibit strongly favorable or unfavorable differential
expression behaviors. A behavior is considered strong if
the ratio of the higher level to the lower level is at least
two-fold. .
' However, a mouse gene may still be identified as
15' favorable or unfavorable even if none of its observed
behaviors arestrong as defined above. In general, we
consider the consistency of its behaviors (that is, are all
or most of the differential expression behaviors at
different ages. in the same direction, e.~g., hyperinsulinemic
20 higher than control), the magnitude of the behaviors (higher
the better), and the expression behavior of structurally or '
functionally related mouse genes (a mouse gene is more
likely to be identified as favorable on the basis of a
weakly favorable behavior. if it is related to other mouse
25 genes which exhibited, favorable, especially strongly
' favorable, behavior). If we~considered a mouse gene with '
only weak differential expression behavior to be worthy of
consideration on the. basis~of these criteria, then we listed
it in Master Table 1 i.n the appropriate subtable.
Preferably, the differential behavior observed is both
strong and consistent_=Preferably, if related mouse genes
;' were tested, they exhibit the'same direction of differential
expression.behavior.
' 35 . ' A mouse gene which was more strongly expressed in~
,hyperinsulinemic. tisst.~.e than iri either normal or type hI '
diabeti~;tissue~(i.e., C<HI, HI>D) will be deemed both
'~ "unfavorable", by virtue of the control,:hyperinsulinemic, '
cornparison,~. and ,"'favorable", by virtue of~ the ~ '
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26
hyperinsulinemic:diabetic comparison. This is one of several
possible "mixed" expression patterns..
Thus, we can subdivide the "favorables" into wholly and
partially favorables. Likewise, we can subdivide the
unfavorables into wholly and partially unfavorables. The
genes/proteins with "mixed" expression patterns are, by
definition, both partially favorable and partially
unfavorable. In general, use of the wholly favorable or
wholly unfavorable genes/proteins is preferred to use of the
partially favorable or part Tally unfavorable ones.
It is evident from the foregoing that mixed
genes/proteins are those exhibiting a combination of
favorable and unfavorable behavior. A mixed gene/protein
can be'used as would a favorable gene/protein if its
favorable behavior outweighs the unfavorable. It can be
used as would an unfavorabl a gene/protein if its unfavorable
behavior outweighs the favorable. Preferably, they are used
in conjunction with other agents that affect their balance
of favorable and unfavorabl a behavior: Use of mixed
genes/proteins is, in general, less desirable than use of
purely favorable or purely unfavorable.genes/proteins, but
it is not excluded.
It should be noted that a mouse gene is classified on
'the basis of the strongest C-HI behavior among the ages
tested, the strongest HI=D behavior among the ages tested,
and the strongest C-D behavior among the ages tested. If at
least one of these three,bahaviors is significantly
favorable, and none of the others of these~three behaviors
is significantly unfavorabl e, the mouse gene will be
classified as wholly favorable~and listed in subtable 1.A of
Master Table 1. However,, t hat does not mean that it may not
have exhibited~a weaker but unfavorable expression'behavior
at some tested age.
The "favorable" , "unfavorable" and "mixed" mouse ,
proteins of the present'invention include the mouse database
proteins listed in the Mast er Table in the same row as a
particular "favorable",'"unfavorable" or "mixed" mouse gene,
;respectively.. These proteins may be the exact translation v
productwof the identified mousegene (database DNA) .
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27
However, if they were 'sequenced.directly, they could be
shorter or longer than that translation product. They could
also differ in sequence from the exact translation product
as a result of post-translat Tonal modifications.
The mouse proteins of interest also include mouse
proteins which, while not listed in the table, correspond to
(i.e., homologous to, i.e., which could be aligned in a
statistically significant manner to) such mouse proteins or
genes, and mouse proteins which are at least substantially
identical or conservatively identical to the listed mouse
proteins.
Related human genes (database DNAs) and proteins were
identified by searching a database comprising human DNAs or .
proteins for sequences corresponding to (i.e., homologous
to,' i.e., which could be aligned in a statistically
significant manner to) the mouse gene or protein. More than
one human protein may be.identified as corresponding to a
particular mouse chip probe and~to a particular mouse gene.
Note that the terms "human genes" . and "human proteins"
are used in a manner analogous to that already discussed in
the case of ;"mouse genes" and ."mouse proteins" .
As used herein, the term "corresponding" does not mean
identical, but rather implies the existence of,a
statistically significant sequence similarity, such as one
sufficient,to qualify the human protein or gene as a
hornologus protein or DNA as defined below. The greater the
degree of relationship ,as thus def fined ( i . a . , by the
statistical significance of each alignment used to connect
the mouse cDNA to the human protein or gene, measured by an
E value),~the more close the correspondence. The connection
may be;direct (mouse gene to human p~otein)'or indirect
(e. g., mouse gene to human gene, human. gene to. human
protein)~.By "mousevgene", we mean the mouse gene from which '
the gene chip DNA ,in question was derived. '. , ,
In general, the human ganes/proteins which,most closely
correspond, directly or indirectly; to the mouse genes are.
,;
,preferred,. such a's the one (s) with the highest, top two
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28
highest, top three highest, top four highest, top five
highest, and top ten highest E values for the final
alignment in the connection process. The human
genes/proteins deemed to correspond to our mouse genes are
identified in the Master Tables.
Note that it is possible to identify homologous full-
length human genes and proteins, if they are present in the
database, even if the query mouse DNA or protein sequence is
not a full-length sequence.
If there is no homologous full-length human gene or
protein in the database, but there is a partial one, the
latter may nonetheless be useful. For example, a partial
protein may still have biological activity, and a molecule
which binds the partial protein may also bind the full-
.length protein so as to antagonize a biological activity of
the 'full-length protein. Likewise, a partial human gene may
encode a partial protein which has biological activity, or
the gene may be useful in the design of a hybridization
probe or in the design of a therapeutic antisense DNA.
The partial genes and protein sequences may of course
also be used in the design of~probes intended to identify
the full length gene or protein sequence.
For the sake of convenience, we refer to a human
protein as favorable if (1) it is listed in Master Table 1
as corresponding to a favorable mouse gene, or (2) it is at
least substantially identical or conservatively identical to
a listed protein per (1),:or (3) it is a member of a human
protein class listed in Master Table 2 (if,provided) as
corresponding,to a favorable mouse gene. We define a human v
protein as unfavorable in an analogous manner. We may
further identify a human protes..n as being wholly,,favorable~~
(see mouse genes of subtable 1A, ,wholly unfavorable (see
mouse genes of subtable 1B), or mixed, i.e., both, partially
favorable and partially unfavorable (see mouse'' genes of ,
subtable 1C).
Likewise, a human gene which encodes a.~particular human
protein may be classi fled in th.e same way as the human
protein which it encodes:
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29
However, it should be noted t hat this classification is
not based on the direct study of t he expression of the human
gene/protein. of course, the human genes/proteins of
ultimate interest will be the ones whose change in level of
expression is, in fact, correlated, directly or inversely,
with the change of state (normal,.hyperinsulinemic,
diabetic) of the subject.
After identifying related human genes and proteins, one
may formulate agents useful in screening humans at risk for.
pr-ogression toward hyperinsulinemia or toward type II
diabetes, or protecting humans at risk thereof from
progression from a normoinsulinemic state to a
hyperinsulinemic state, or from either to a type II diabetic
'15 state.
Agents which bind the "favorable" and "unfavorable"
nucleic acids (e. g., the agent is a substantially
complementary nucleic acid hybridization probe), or the
corresponding proteins (e. g., an antibody vs. the protein)
may be used to evaluate whether a human subject is at
increased or decreased risk for progression toward type II
diabetes. A subject with one or more elevated "unfavorable"
and/or one or more depressed "favorable" genes/proteins is
at increased risk, and one with one or more elevated '
"favorable" and/or one or more depressed "unfavorable"
genes/proteins is at decreased risk. ~ne may further take
into account whether the subject is normoinsulinemic or
hyperinsulinemic at the time of the assay. If the subject ,
is non-diabetic and normoinsulinem3.c, we are especially , ,
interested in the "favorable" and "unfavorable " human
genes/proteins corresponding to mouse genes differentially
expressed in hyperinsulinemic vs. normal muscle. If the
subject~is already hyperinsulinemic, yet non-diabetic, we
35~ are especially interested in the "favorable" ands '
"unfavorable" human genes/proteins corresponding to,mouse ,'~
genes differentially expressed in type II diabetic vs.
hyperinsulinemic, muscle.
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The assay may be used as a pre liminary screening assay
to select subjects for further analysis, or as a formal
diagnostic assay.
5 The identification of the related genes and proteins
may also be useful in~protecting humans against these
disorders. . ~ ' '
Thus, Applicants contemplate:
(1) use of the "favorable" mouse DNAs (or fragments
10 thereof) o'f the Master Tables (below) to isolate or identify
related human DNAs;
(2) use of human DNAs, related to favorable mouse DNAs,
to express the corresponding,human proteins;
_ (3) use of the corresponding human proteins, (and mouse
15 ~proteins,_if biologically active in. humans), to protect
against the disorder(s);
(4) use of~the corresponding mouse or human proteins,
or nucleic acid probes derived from the mouse or human
genes, in diagnostic agents, in ass ays to measure
20 progression toward hyperinsulinemia or type II diabetes, or
protection against the' disorder(s), or to estimate related
end organ damage such as kidney damage; and
(5) use of the corresponding human or mouse genes
therapeutically in gene therapy, to protect against the
2.5 diBorder (s) .
Moreover Applicants contemplat el:
(1) use of the "unfavorable" mouse DNAs (or fragments
thereof) of the Master.Tables'to isolate or identify related
human DNAs; . ~ '
30 (2) use of the complement to t he "unfavorable" mouse
DNAs or related human DNAs, asvantisense molecules to '
inhibit expression of the related human DNAs;
(3) use of the.mouse or human DNAs to express the
corresponding,mouse,or human proteins; ,
' (4) use of the corresponding'mouse or human proteins,
in diagnostic agents, to measure progression toward
:,hyperinsulinemia or type II diabete s,~or protection against
' the disorder(s)., or to estimate related end organ damage
such as, kidney damage;, ~ ~ ~ '
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(5) use of the corresponding mouse or human proteins in
assays to determine whether a substance binds to (and hence
may neutralize) the protein; and
(6) use of the neutralizing substance to protect
against the disorder(s).
Thus, DNAs of interest include those which specifically
hybridize to the aforementioned mouse or human genes, and
are thus of interest as hybridization assay reagents or for
antisense therapy. They also include synthet is DNA sequences
which encode the same polypeptide as is encoded by the
database~DNA, and thus are useful for producing the
polypeptide in cell culture or in situ (i.e., gene therapy).
Moreover, they include DNA sequences which encode
polypeptides which are substantially structurally identical
or conservatively identical in amino acid sequence to the
mouse and human proteins identified in'the Master Table 1,
subtables 1A or 1C. Finally, they include DNA sequences
which encode peptide (including antibody) antagonists of the
proteins of Master Table 1, subtables 1B or 1C.
The related human DNAs may be identified by comparing
the mouse sequence (or its AA translation product) to known
human DNAs (and their AA translation products) .
25,. Related human DNAs also may be identif=i.ed by screening
human cDNA or genomic DNA libraries using t:he mouse gene of
the Master Table, or,a fragment thereof, as a probe.
If the,mouse gene of Master Table 1 is not full-length, and
there is no closely corresponding full-length mouse gene in
the sequence databank, then the mouse DNA may first be used
~,as a hybridization probe to screen a mouse cDNA library to
isolate the corresponding full-length sequence.
Alternatively,~the mouse DNA may be used as a probe to
. . screen a mouse genomic DNA library. , ,
Our animal models of hyperinsulinemia and diabetes are
also obese. It is possible that~the genes found to be.
favorable act indirectly by inhibiting~obe~ ity...Likewise, it,
is possible that the ~~.genes~~found~ to bel unfavorable act I
indirectly by accentuating obesity. Consequeritly, it~ is
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within the compass of the present invention to use the
favorable genes and proteins, or to use antagonists of the
unfavorable genes and proteins, to protect against obesity,
as well as against sequelae of obesity such as
hyperinsulinemia and diabetes.
Since type II diabetes is an age-related disease, th.e
agents of the present invention may be used in conunction
with known anti-aging or anti-age-related disease. agents.
It is of particular interest to use the agents of the
l0 present invention in conjunction with an agent disclosed.in
one of the related applications cited above, in particular,
an antagonist to CIDE-A, the latter having been taught in
Kopchick7 and Kopchick7A-PCT.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Body weight gain [Fig. 1a], fasting glucose [Fig.
1b] and fasting insulin [Fig. 1c] levels of mice on the HF
or Std diets.
Figure 2. Expression levels of Actin, alpha, cardiac
(Actcl, NM 009608) using RNA isolated from gastrocnamius
muscle of individual diabetic HF mice and corresponding Std
mice at different time points.
'
Figure 3. Data shown are expression levels for addit Tonal
actin-related and actin-binding genes exhibiting a
consistent decrease in expression in the HF mice in
comparison to Std mice at all four time points (Fig_ 3(a))'
or at three of the four time points (Fig. 3(b)).
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
Full-Length ors. Partial Length Genes/Proteins
A "full length" gene is here defined as (1) a
naturally occurring DNA sequence which begins with an
initiation codon (almost alwaysthe Met codon, ATG), and
ends with a stop codon in phase with said initiation c odon
(when introns, if any, are ignored), and thereby encodes 'a
naturally occurring polypeptide with biological activity, or
a naturally occurring precursor thereof, or (2) a synthetic
DNA sequence which encodes the same polypeptide as that ,
which is encoded by (1). ~ The gene may, but need not,
include introns.
A "full-length"~ protein is here defined as a
naturally occurring protein encoded by a full-length gene,
or a protein derived naturally by post-translational
modification of such a protein. Thus, it includes mat ure
proteins, proproteins, preproteins and preproproteins. It
also includes substitution and extension mutants of such '
naturally occurring proteins..
Subjects '
' A mouse is considered to be a diabetic subject if,
regardless of its fasting plasma insulin level, it has a
fasting plasma glucose level of at least 190 mg/dL. A mouse
is considered to~be a hyperinsulinernic.subject if its
fasting plasma insulin level is at least 0.67 ng/mL and it
does'.not qualify as ~. diabetic subject. A mouse is
considered to be'"normal" if it is neither~diabetic no r~~ ~ .
hyperinsulinemic. Thus,'normality is defined in a very ~ '
limited manner. .
A'mouse is considered."obese" if its weight is at least
' 15a in excess of themean weight for mice of its age and
sex. A mouse which.doesvriot satisfy'this standard may be '
characterized as "non-obese°', the term "normal" being . ,
reserved for use in reference to glucose and insulin 1 evels;
as previously described. ~ ' '
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A human is considered a diabetic subject if, regardless
of his or her fasting plasma insulin level, the fasting '
plasma glucose level.is at least 126 mg/dL. A human is
considered a hyperinsulinemic subject if the fasting plasma
5 insulin level is more than 26 micro International Units/mL
(it is believed that this is equivalent to 1.08 ng/mL), and
does not qualify as a diabetic subject. A human is
considered to be "normal" if it is neither diabetic nor
hyperinsulinemic. Thus, normality is defined in a very
10 limited manner.
A human is considered "obese" if the body mass index
(BMI) (weight divided by height'squared) is at least 30
kg/m2. A human who does not satisfy, this standard may be
characterized as "non-obese", the term "normal" being
15 reserved for use in reference to glucose and insulin level s
as previously described.
A human is considered overweight if the BMI is at lea st
25 kg/m2. Thus, we define overweight to include obese
individuals, consistent with the recommendations of the
20 National Institute of Diabetes and Digestive and Kidney
Diseases(NIDDK). A human who does not satisfy this standard
may be characterized as "non-overweight."
According to the Report of the Expert Committe on the
25 Diagnosis and Classification of Diabetes Mellitus, Diabete s
Care 20: 118397 (1997) , the following are risk factors for
diabetes type II:
older (e. g., at least~45; see below)
'. excessive weigYit '(see below)
. , ,
first-degree. relative with diabetes mellitus
35; member of high risk ethnic group (bl,ack, ~Hispariic,
Native 'American, Asian) ~ ' '
, history of gestational diabetes mellitus,or delivering
a ,baby wea.ghing more .than 9~ pounds (4 . 032 kg)
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hypertensive~(>140/90 mm Hg)
HDL cholesterol level >35 mg/dL (0. 90 mmol/L)
triglyceride level >=250 mg/dL (2.g 3 mmol/L)
Hence, in a preferred embodiment, t=he diagnostic and
protective methods of the present invent=ion are applied to
human subjects exhibiting one or more of= the aforementioned
risk factors. Likewise, in a preferred embodiment, they are
applied to human,subjects who, while not= diabetic, exhibit
impaired glucose homeostasis (110 to <12 6 mg/dL).
The risk of diabetes increases witY3. age. Hence,. in
successive preferred embodiments, the age of the subjects is
at least 45, at least 50, at least 55, ~t least 60, at least
65, at least.70, and at least 75.
'With regard to excessive weight, N~ DDK says that "The
relative risk of diabetes increases by approximately 25
percent for each additional unit of BMI over 22." Hence, in
successive preferred embodiments, the BI~Is of the human
subjects is at least 23, at least 24, at. least 25 (i.e.,
overweight by our criterion), at least 2 6, at least 27, at
least 28, at~least 29, at least 30 (i.e. , .obese), at least
31, at least 32, at least 33, at least 3 4,~ at least 35, at
least 36, at least 37, at least 38, at L east 39, at least
40, or over 40. y
Age-Related Diseases
Age=related (senescent) diseases ir~clude certain
cancers, atherosclerosis, diabetes (type 2), osteoporosis,
hypertension, depression, Alzheimer's, Parkinson's, glaucoma,
certain immune system defects, kidney failure, and liver
steatosis. In general, they are diseases for which the
relative risk (comparing a subpopulatior~ over age 55 to a
suitably matched population,under age 55) is at'~least~ 1.1.
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Preferably, the agents of the present invention protect
against one or more age-related diseases for at least a
subpopulation of mature (post-puberty) adult subjects.
Direct and Indirect Utility. of Identified Nucleic Acid
Sequences and Related Molecules
The mouse or human genes (or fragments thereof) may be
used directly. For diagnostic or,screening purposes, they
(or specific binding fragments thereof) may be labeled and
used as hybridization probes. For therapeutic purposes, ,
they (or specific binding,fragments thereof) may be used as
antisense reagents to inhibit the expression of the
15, corresponding gene, or of a sufficiently homologous~,gene .of
another species.
If the database DNA appears to be a full-length cDNA
or gDNA, that is, it encodes an entire, functional,
naturally occurring protein, then it may be used~~in the
expression of that protein. Likewise, if the corresponding
. human gene is known in full-length, it may be used to ,
express the human protein. Such expression may be in cell
culture,'with the protein subsequently isolated and
administered exogenously to subjects'who would benefit
therefrom, or in vivo, i.e., administration by gene therapy.
Naturally, any DNA encoding the same protein may be used for
the same purpose, and a DNA encoding a protein which a
fragment or a mutant of that'naturall.y occurring protein
which retains the desired activity,..may be used for the
purpose of producing the active fragment or mutant. The ,
encoded protein of course has'', utility therapeutically~and,
in labeled or immobilized form, diagnostically. ;
The genes may also~be used,indirectly,~that is, to
identify other useful DNAs, ~ proteins, for other .molecules.
We have attempted to determine whether the mouse genes
disclosed herein have significant similarity to any.knownw
human DNA, and whether, in any of the six possible 1,
combinations of reference frame and,strand, they encode. a; ,.
protein similar. to ~a known human protein. ~. If .so; then it
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follows that the known,human protein, and DNAs encoding that
protein, may be used in a similar manner. In addition, if
the known human protein is known to have acZditional
homologues, then those homologous proteins, and DNAs
encoding them, may be used in a similar manner.
There thus are several ways that a human protein
homologue of interest can be identified by database v
searching, including but not limited to:
1) a DNA->DNA.(BlastN) search for human dat=abase DNAs
closely related to the~mouse gene identifies a known human
gene, and the sequence of the human protein is deduced by
the Genetic Code;
2) a DNA->Protein (BlastX) search for humn database proteins
closely related to the translated DNA of the mouse gene
identifies a known human protein; and
3) the sequence of the mouse protein is known or is deduced
by the Genetic Code, and a Protein->Protein (BlastP) search
for closely related database proteins ident=ifies a known
human protein.
Qnce a known human gene is identified, it rnay be used in
further BlastN or BlastX searches to ident3 fy other human
,genes or proteins. Once a known human protrein is ~ '
identified, it may be used in further BlastrP searches to~, ,
identify other human proteins..
Searches may also take. cognizance, intermediately, of known
genes and proteins other than mouse or human ones e.g.; use
the mouse sequence to identify a known rat sequence and then
the rat sequence to identify a human one.
If we have identified a mouse gene, and it encodes a
mouse protein which appears similar to a hurrian protein, 'then,
that human protein may be used (especially inwhumans) for
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purposes analogous to the proposed use of the mouse protein
in mice. Moreover, a specific binding fragment of an
appropriate strand of the corresponding human gene (gDNA or
cDNA) could be labeled and used as a hybridization probe
(especially against samples of human mRNA or cDNA).
In determining whether the disclosed genes (gDNA or
cDNA)have significant similarities to known DNAs (and their
translated AA sequences to known proteins), one would
generally use the disclosed gene as a query sequence in a
search of,a sequence database. The results of several such
searches are set forth in the Examples. Such results are
dependent, to some degree, on the search parameters.
Preferred parameters are set forth in Example 1. The
results'are also dependent on the~content of the database.
While the raw similarity score of a particular target
(database) sequence will not vary with content (as.long as
it remains in the database), its informational value (in
bits), expected value, and relative ranking can change.
Generally speaking, the changes are small.
It will be appreciated that the nucleic acid and
protein databases keep growing. Hence a later search may
identify high scoring target sequences which were not
uncovered by an earlier search because the target sequences
,were not previously part of a database.
Hence, in a preferred embodiment, the cognate DNAs.and
proteins include riot only those set forth in the examples,
but those which would have been highly ranked (top. ten, more
preferably top three, even more preferably top two,,most ,
preferably the top one) in a search run with the same
parameters on the date of filing of this application .,
If the known..mouse or human database DNA appears to be
a partial sequence (that is;,~partial relative to a cDNA or
gDNA encoding the whole naturally occurring protein), it may
be used, as al, hybridization probe to isolate the full-length
DNA. If.the.partial DNA encodes a biologically functional.
fragmentrof the cognate protein, lit may be used in a manner.
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similar to the full length DNA, i.e., to produce the
functional fragment. .
If we have indicated that an antagonist of a protein or
5 other molecule is useful, then such an antagonist may be
obtained by preparing a combinatorial library, as described
belqw, of potential antagonists, and screening the library
members for binding to the protein or other molecule in
question. The binding members may then be further screened
10 for the ability to antagonize the biological activity of the
target. The antagonists may be used therapeutically, or, in
suitably labeled or immobilized form, diagnostically.
If the identified mouse or human database DNA is
related to a known protein, then,substances known to
15 interact with that protein (e.g.agonists, antagonists,
substrates, receptors, second messengers, regulators, and so
forth), and binding molecules which bind them, are also of
utility. Such binding molecules can likewise be identified
by screening a combinatorial library.
Isolation of Full Length DNAs Using Partial DNAs as probes
If it is determined that a DNA of the present invention
is a partial DNA, and the cognate full length DNA is not
,listed in a sequence database, the available DNA may be used
as a hybridization probe to isolate the full-length DNA from
a suitable DNA library.
Stringent.hybridization.conditions are appropriate,
.that is, conditions in which the hybridization temperature
is 5-10 deg. C. below the Tm of, the DNA as a~perfect duplex.
Te3entification and Tsolation~of Homologous Genes Using a DNA
Probe . ..
It may be that the sequence databases available do not
include the sequence of any homologous gene (cDNA or gDNA), ,
or at least of the homologous gene for a species of
. : interest. However, given,the cDNAs set forth above, one'may '
readily obtain the homologous;,gene.
. ~ The possession of 'one DNA (the "starting DNA" ) greatly
,faci~litateswthe isolation of homologous DNAs. If only a
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partial DNA is known, this~partial DNA may first be used as
a probe to isolate the corresponding full length DNA for the
same species, and that the latter may be used as the
starting DNA in the search for homologous genes.
The starting DNA, or a fragment thereof, is used as a
hybridization probe to.screen a cDNA or genomic DNA library
for clones containing inserts which encode either the entire
homologous protein, or a recognizable fragment thereof. The
minimum length of the hybridization probe is dictated by the
need for 'specificity. If the size of the library in bases
is L, and the GC content is 50%, then the probe should have
a length of at least l, where L = 41. This will yield, on
average, a single perfect match in random DNA of L bases.
The human cDNA library is~ about 10$ bases and the human
genomic DNA library is about 101° bases.
The library is preferably derived from an organism
which is,known, on biochemical evidence, to produce,a
homologous protein, and more preferably from the genomic DNA
or mRNA of cells of that organism which'are likely to be
relatively high producers of that protein. A cDNA library
(which is derived from,an mRNA library) is especially ~ ,
preferred.
If the organism in question is known to have
substantially different~codon preferences from that of the
organism whose'relevant cDNA or genomic DNA is known, a
synthetic hybridization probe may be used which encodes the
same amino acid sequence but whose codon utilization is more
similar.to that~of the DNA of the,target organism. , . '
Alternatively, the synthetic probe may employ inosine as a
substitute for those bases which are most likely.to be ''
' divergent, or~the probe may be a mixed probe which mixes the '
..
codons for;the source DNA with the preferred codons '
(encoding the same amino acid) for the target organism.
By routine methods,.the Tm of a perfect duplex of ~ ' '
starting DNA is determined.' One may then select a
.hybridization temperature which is sufficiently~lower than ~v
the perfect duplex Tm to allow~vhybridization;of the starting
DNA. (or other probe) to ~ a .target DNA which i\s divergent from
the, starting ;DNA. A 1% sequence divergence' typically lo~,vers ; ' .~ ,
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the Tm of a duplex by 1-2°C, and the DNAs encoding
homologous proteins of different species typically have
sequence identities of around 50-800. Preferably, the
library is screened under conditions where the temperature
is at least 20°C., more preferably at least 50°C., below the
perfect duplex Tm. Since salt reduces the Tm, one
ordinarily would carry out the search for DNAs encoding
highly homologous proteins under relatively low salt
hybridization conditions, e.g., <1M NaCl. The higher the
salt concentration; and/or the lower the temperature, the
greater the sequence divergence which is tolerated.
For the use of probes to identify homologous genes in
other species, see, e.g., Schwinn,.et al., J. Biol. Chem.,
265:8183-89 (1990) (hamster 67-by eDNA probe vs. human
115 leukocyte genomic library; humaw0.32kb DNA probe vs. bovine'
brain cDNA library, both with hybridization at 42°C in
6xSSC); Jenkins et al., J. Biol. Chem., 265:19624-31 (1990)
(Chicken 770-by cDNA probe vs. human genomic libraries;
hybridization at 40°C in 50% formamide and 5xSSC); Murata et
al., J. Exp. Med., 175:341-51 (1992) (1.2-kb mouse cDNA
probe v. human eosinophil eDNA library; hybridization at
65°C in 6xSSC); Guyer et al., J. Biol.,Chem., 265:17307-17
(1990) (2.95-kb human genomic DNA probe vs. porcine genomic
DNA library; hybridization at'42°C in 5xSSC). The
conditions set forth. in these articles may each be
considered suitable for the purpose of isolating homologous
genes.
Correspondir~.g (Homologous) Proteins and DNAs
In the case of a gene chip, the manufacturer of the gene
chip determines which DNA to place at each position on the
chip. This DNA may correspond in. sequence to a genomic DNA,
a cDNA, or a fragment of genomic or, cDNA, and may be
natural, synthetic or partially~riatural and partially .
synthetic in'origin. The~manufacturer of the gene chip'will
normally identify the'DNA for a mouse'gene;chip as '
corresponding to a particular mouse gene, in which.cas,e it
will be assumed that the alignments olf~chip'DNA to mouse
,gene satisfies the homology criteria of the ;invention.
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Usually, the gene chip manufacturer willprovide a sequence
database accession number for the mouse DNA. If so, to
identify the corresponding mouse protein, we will first
inspect the database record for that mouse DNA. Often, the
mouse protein accession number will appear in that record or
in a linked record. If it doesn't, the corresponding mouse.
protein can be identified by performing a BlastX search on a
mouse protein database with the mouse database DNA sequence
as the query sequence. Even if the protein sequence is not
in the database, if the DNA sequence comprises a full-length
coding sequence, the corresponding protein can be identified
by translating the coding sequence in accordance with the
Genetic Code.
A human protein can be said to be identifiable as
corresponding (homologous) to a gene chip DNA if it is
identified as corresponding (homologous) to the mouse gene
(gDNA or cDNA, whole or partial) identified by the gene chip
manufacturer as corresponding to that gene chip DNA.
In turn, it is identifiable as corresponding
(homologous) to said identified mouse gene, if
(1) it can be aligned by BlastX directly to that mouse gene,
and/or
(2) it is'encoded by a human gene, or can be aligned to a
human gene,by BlastX, which in turn can be aligned by BlastN
to said~mouse gene and/or
(3) it can be. aligned by BlastP to a mouse protein, the '. ',
latter being encoded by said mouse gene, or aligned to; said
mouse gene BlastX,
where any alignment by BlastN,' BlastP or BlastX is in
accordance'with the default parameters set,,forth below, and
,the expected value '(E) of each alignment,(the probability
' that such an alignment would have occurred by chance~~al'one)'
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is less than e-10. (Note that because this is a negative
exponent, a value such as e-50 is less than e-10.)
Desirably, two or all three of these conditions (1)-(3) are
satisfied for the corresponding (homologous) human genes and
proteins.
A human gene is corresponding (homologous) to a mouse
gene chip DNA, and hence to said identified mouse gene (or
cDNA) and protein, if it encodes a corresponding
(homologous) human protein as defined above, or it can be
aligned by BlastN to said mouse gene.
Preferably, for at least one of conditions (1)-(3), the
E value is less than e-50, more preferably less than e-60,
still more preferably less than e-70, even more preferably
less than e-80, considerably more preferably less than e-90,
and most preferably less than e-100. Desirably, it is true
for two or even all three of these conditions.
In constructing Master table l, we generally used a
BlastX (mouse gene vs. human protein) alignment E value
cutoff of e-50. However, if there were no human proteins
with that good an alignment to the mouse DNA in question, or
if there were other reasons for including a particular human
protein'(e.g., a known functionality supportive of the
observed differential cognate mouse protein expression),
then a human protein with a score worse (i.e., higher) than
e-50 may appear in Master Table 1:.
30' If the manufacturer of the gene chip identifies the
gene chip DNA aS corresponding ,to an EST, or other DNA which
is not; a full-length mouse gene or cDNA, 'a longer (possibly
full length) mouse gene.or cDNA maybe identified by a
BlastN search of the mouse DNA database. Alternatively, the
identified DNA'may be used to conduct a BlastN search of a
human DNA.database, or a BlastX search of ~a mouse or human
protein ~databa'se .
Thus'; more'generally, a human protein can be said to be
identifiable as corresponding (homologous) to a gene chip
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DNA, or to a DNA identified by the manufacturer as
corresponding to that gme chip DNA, if
(1') it can be aligned directly to the gene chip or
5 corresponding manufacturer identified DNA by BlastX. and/or
(2') it can be aligned to a human gene/cDNA by BlastX, whose
genomic DNA (gDNA) or cDNA (DNA complementary to messenger
RNA) in turn can be aligned to the gene chip or
10 corresponding manufacturer identified DNA by BlastN, and/or
(3') it can be aligned to a mouse gene/cDNA by BlastX, whose
gDNA or cDNA in turn can be aligned to the gene.chip or~
corresponding manufacturer identified:DNA by BlastN, and/or , '
15 '
(4') it can be aligned to a mouse protein by BlastP, which
in turn can be aligned to the gene chip or corresponding
manufacturer identified DNA by BlastX, and/or
20' (5') it can be aligned to a mouse protein by BlastP,,which
in turn can be aligned to a mouse gene/cDNA by BlastX, whose
gDNA or cDNA can in turn be aligned to the gene chip or
corresponding manufacturer identified DNA by BlastN;
25 where any alignment by BlastN, BlastP; or BlastX is in
accordance with the default parameters setforth below, and
the expected value (E),of each alignment (the probability
that such an.alignmerit would have occurred by chance alone) '
is less than e-10. (Note that because this is a negative
30 exponent; a value such as e-50 i,s Less than e-10.)
. , ,
Preferably, two, , three; ~ four or all five of conditions
(1' ) - (5' ) are satisfied: ,
Preferably, for at least one of conditions (1' ) - (5' ) ,
35 for at least the final alignment (i.e.; vs.',the human
protein), the E value is less than e-50, more preferably ~ w
less than e-60, ;;still more preferably less than e-70, even ,
more preferably less than e-80; considerably more'preferably
less than e=.90, and most preferably less. than e-100.
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46
Desirably, one or more of these standards of preference
are met for two, three, four or all five of conditions (1')-
(5'). In particular, for those conditions in which the gene,
chip or corresponding manufacturer identified DNA is.
indirectly connected to the human protein by virtue of two
or more successive alignments, the E value is preferably, so .
limited for all of said alignments in the connecting chain.
A human gene corresponds (is homologous) to a gene chip
DNA or manufacturer identified corresponding DNA if it
encodes a homologous human protein as defined above, or if
it can be aligned either directly to that DNA, or indirectly
through a mouse gene which can be aligned to said DNA,
according to the conditions set forth above.
, ,
Master table 1 assembles a list of human protein
corresponding to each of the mouse DNAs/proteins identified
as related to the, chip DNA. These human proteins form a set
and can be given a percentile rank, with respect to E value,
within that set. The human proteins of the present
invention preferably are those scorers with a percentile.
rank of at least 500, more preferably at least 60%, still,
more preferably at least'70%, even more..preferably at least
800, and most preferably at least~90o. '
For each mouse gene (gDNA or cDNA) in Master Table 1, ' w
there is a particular. human protein which provides the best
alignment match as measured by BlastX, i.e., the human
protein with the best score (lowest e-value). These human
proteins form a subset of the set above 'and can be given a
percentile rank~with.in that subset, a.g., ',the human proteins .
with scores in the,top 10% of that subset have a percentile
rank of 90% or higher.
The human proteins of the,present invention preferably
are those~best scorer subset proteins with a percentile rank;
withimthe subset of at, least ~ 50 0, more preferably ..at~ least
60%, still more preferably at least 70%~, even more
preferably at least ~ 80 0 ,, and most preferably at ~ least 90% . .
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BlastN and BlastX report very low expected values as
"0.0". This does not truly mean that the expected value is
exactly zero (since any alignment could occur by chance),
but merely that it is so infinitesimah that it is not
reported. The documentation does not state the cutoff
value, but alignments with explicit E values as low as e-178
(624 bits) have been reported as nonzero values, while a
score of 636 bits was reported as "0.0"
Functionally homologous human proteins are also of
interest. A human protein may be said to be functionally
homologous to the mouse gene if the human protein has at
least one biological activity in common with the mouse
protein encoded by said mouse gene.
The human proteins of interest also include those that
are substantially and/or conservatively identical (as
defined below) to the homologous and/or functionally
homologous human proteins defined above.
Degree of Differential Expression
The degree of differential expression may be expressed
as the ratio of the higher expression level to the lower
expression level. Preferably, this is at least 2-fold,, and
more preferably, it is higher, such as at least 3-fold, at
,25 least,4-fold, at least 5-fold, at,least 6-fold, at,least 7-'
fold, at 7.east 8-fold, at least 9-fold, or at least 10-fold.
~~ Most preferably, the human protein of interest corresponds
to a mouse gene for which the degree of differential
expression places it among the top 100 of the~mouse genes in
the appropriate subtable.
Relevance of Favorable and Unfavorable Genes
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If a gene is down-regulated in more,favored mammals, or
up-regulated in. less favored mammals, (i.e., an "unfavorable
gene") then several utilities are apparent.
First,,the complementary strand of the gene, or a
portion thereof, may be used in labeled form as a
hybridization.probe to detect. messenger RNA and thereby
monitor the level of expression of the gene in a subject.
Elevated levels are indicative of progression, or
propensity to progression, to a less favored state, and
clinicians may take appropriate preventative, curative or
ameliorative action.
Secondly; the messenger RNA product (or equivalent
cDNA), the protein product, or a binding molecule specific
for that product (e.g., an antibody which binds the
product), or a downstream product which mediates the
activity (e. g., a signaling intermediate) or a binding
molecule (e. g., an antibody) therefor, may be used,
preferably in labeled or immobilized form, as an assay
reagent in an assay for said nucleic acid product, protein
product, or downstream product (e. g., a signaling
intermediate). Again, elevated levels are indicative of a
present or future problem.
Thirdly, an agent which down-regulates expression of
the gene may be used to reduce levels of the corresponding
protein and thereby inhibit further damage. This agent
could inhibit transcription of the gene in the subject, or
translation of the corresponding messenger RNA. Possible
inhibitors of transcription and translation include
antisense molecules and repressor molecules. The agent
could also inhibit a post-translational modification (e. g.,
,glycosylation, phosphorylation, cleavage, GPI attachment) '
'required for activity, or post-translationally modify the
protein so as to inactivate it. Or it could be an agent
which down- or up-regulated a positive or negative
regulatory gene, respectively.
Fourthly, an agent~whiCh~is an antagonist of. the
messenger RNA product or protein product of the gene,'or of
a~downstream product through which its.activity,is
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' manifested (e.g., a signaling intermediate), may be used to
inhibit its activity. .
This antagonist could be an antibody, a peptide, a
peptoid, a nucleic acid, a peptide nucleic acid (PNA)
oligomer, a small organic molecule of a kind for which a
combinatorial library exists (e.g.~, a benzodiazepine), etc.
An antagonist is simply a binding molecule which, by
binding, reduces or abolishes the undesired activity of its
target. The antagonist, if not an oligomeric molecule, is
preferably less than 1000 daltons, more preferably less than
500 daltons.
Fifthly, an agent which degrades, or abets the
degradation of, that messenger RNA, its protein product or a
downstream product which mediates its activity ~(e.g., a
signaling intermediate), may be used to curb the effective
period of activity of the protein.
If a gene is ,u~,-regulated in more favored mammals, or
down-regulated in less favored animals then the utilities
are converse to those stated above. '
First, the complementary strand of the gene, or a
portion thereof, may be used in labeled form as a
hybridization probe to detect messenger RNA and thereby
monitor the level of expression of the gene in a subject.
Depressed levels are indicative of damage, or possibly of a
propensity to damage, and clinicians may take appropriate
preventative, curative or ameliorative action.
Secondly, the messenger RNA product,. the equivalent
cDNA, protein product,.or a binding molecule specific for
those products, or a downstream~product, or a signaling
intermediate, or a binding molecule therefor, may be used,
preferably .in~labeled or'immobilized form, as an assay
reagent in an assay for said protein product or downstream ,
product.' Again, depressed levels are indicative of a
present or future problem. ,
, ; ' Thirdly,' an agent 'which ,up-regulates expression of the
gene may be used to increase levels of the corresponding
protein and thereby inhibit further progression to a less
favored state. By way of,example, it could be a,vector
which. carries a copy of the 'gene,, but which expresses the
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gene at higher levels than does the endogenous expression
system. Or it could be an agent which up- or down-regulates
a positive or negative regulatory gene.
Fourthly, an agent which is an agonist of the protein
5 product of the gene, or of a downstream product through
which its activity (of inhibition of progression to a less
favored state) is manifested, or of a signaling intermediate
may be used. to foster its activity.
Fifthly, an agent which inhibits the degradation of
10 that protein product or of a downstream product or of a
signaling intermediate may be used to increase the effective
period of activity of the protein.
15 Mutant Proteins
The present invention also contemplates mutant proteins
(peptides) which are substantially identical (as defined
below) to the parental protein (peptide). In general, the
fewer the mutations, the more likely the mutant protein is
20 t o retain the activity of the parental protein. The effect
of mutations is usually (but not always) additive. Certain
individual mutations are more likely to be tolerated than
others .
A protein is more likely to tolerate a mutation which
25 (a) is a substitution rather than an insertion or
deletion;
(b) is an insertion or deletion at the terminus ,
rather than:internally, or, if internal, is at a
domain boundary, or a loop or turn, rather~than in
30 , an alpha ri.elix or .beta strand; ,
(c) aff~ets a surface residue rather than an
interior residue;,
(d) affects a part of the molecule distal to the
binding site;
35' (e) is a,substitution of.one amino acid for
another of similar size,.charge, and/or
hydrophobicity,~.and does not' destroy a disulfide's_
' bond ~or other crosslink; and ~,;
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(f) is at a site which is subject'to substantial
variation among a family of homologous proteins to
which. the protein of interest belongs.
These considerations can be used to design functional
mutants.
Surface vs. Interior Residues
Charged amino acid residues almost always lie on the
surface of the protein. For uncharged residues, there is
less certainty, but in general, hydrophilic residues are
partitioned to the surface and hydrophobic,residues to the
interior. Of course, for a membrane protein, the membrane-
spanning segments are likely to be rich in hydrophobic
residues.
Surface residues may be identified experimentally by
various labeling techniques, or by 3-D structure mapping
techniques like X-ray diffraction and NMR. A 3-D model of a
homologous protein can be helpful.
Binding Site Residues
Residues forming the binding site may be identified by
(1) comparing the effects of labeling the surface residues
before and after complexing the protein to its target, (2)
labeling the binding site directly. with affinity ligands,
(3)' fragmenting the protein and~testing the fragments for
binding activity, and ,(4) systematic mutagenesis (e.g.,
alanine-scanning mutagenesis.) to determine which mutants
destroy binding. If the binding~site of a homologous
protein is known, the binding site may be,postulated by
analogy. , ,,
Protein libraries may be constructed and screened that
a large family (e.g. , 108) of related mutants may; be
evaluated simultaneously. , ~.
Hence, the mutations are preferably conservative
modifications as defined below.
"Substantially.Identical" .
A mutant protein (peptide). is substantially identical.
to a reference protein (peptide) ' ,if (a) it, ,has at least ,10 0
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of a specific binding activity or a non-nutritional , ,
biological activity of the reference protein, and (b) is at
least 50o identical in amino acid sequence to the reference
protein (peptide). 2t is "substantially structurally
identical" if condition (b) applies, regardless of (a).
' Percentage amino acid identity is determined by
aligning the mutant and reference sequences according to a
rigorous dynamic programming algorithm which globally aligns
their sequences to maximize their similarity, the similarity
being scored as the sum of scores for each aligned pair
according to an unbiased PAM250 matrix, and a penalty for
each internal gap of -12 for the first null of the gap and -
4 for each additional null of the same gap. The percentage
identity~is the'number of matches expressed as a percentage
of the adjusted (i.e., counting inserted nulls) length of
the reference sequence.
A mutant DNA sequence is substantially identical to a
reference DNA sequence if they are structural sequences, and
encoding mutant and reference proteins which are
substantially identical as described above.
If instead they are regulatory sequences, they are
substantially identical if the mutant sequence has at least
100 of the regulatory activity of the reference sequence,
and is at least 50% identical in nucleotide sequence to the
reference sequence. Percentage identity is determined as
' for proteins except that matches are~scored +5, mismatches -
4, the gap open penalty is -12, and the gap extension
penalty, (per additional null) is -4. ~ .
More preferably, the sequence is not merely
substantially identical but rather is at, least 510, at leastv
66%, at least 75%, at least 80%, at least 85%, at least.
900, at least 95%, at least 96%, at least 97%, at least ,
98% or at least 99% identical in sequence to the reference
sequence. ,
DNA sequences may also be considered "substantially ,
identical" if they hybridize to each other under~stringent,
conditions,~i.a., conditions at which~the Tm of the , ~ ,,
,heteroduplex of the one strand~of the mutant DNA and;the . , ,
more complementary strand of the reference'DNA is,not;in '.
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excess of 10°C. less than the Tm of the reference DNA
homoduplex. Typically this will correspond to a percentage
identity of 85-90%.
"Conservative Modifications"
"Conservative modifications" are defined as
(a) conservative substitutions of amino acids. as
hereafter defined; or
(b)~ single or multiple insertions (extension) or
deletions (truncation) of amino acids at the
termini. .
Conservative modifications are preferred to other ,
modifications. Conservative substitutions are preferred to
other conservative modifications.
"Semi-Conservative Modifications" are modifications
which are not conservative, but which are (a) semi-
conservative substitutions as hereafter defined; or (b)
single or multiple insertions or deletions internally, but
at interdomain boundaries, in loops or in other segments of
relatively high mobility. Semi-conservative modifications
are preferred to nonconservative modifications: Semi-
conservative substitutions are preferred to other semi-
conservative modifications.
Non-conservative substitutions are preferred to other
non-conservative modifications.
The term "conservative° is used here in an alpriori
sense;.i.e., modifications which would be expected to
preserve~3D structure and activity, based on analysis of ;the'
naturally occurring families of homologous proteins and of
past experience with the effects of deliberate mutagenesis,
rather than post facto, a modification already known to
conserve activity. Of course, a modification which is
conservative a priori may, and usually is, also conservative
post 'facto.
~35 Preferably,y.except at the termini, no more than about
five,amino acids are inserted or deleted~at~a.particular:
locus, and the modifications are outside regions known to
contain binding sites important to activity.
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Preferably, insertions or deletions are limited to the
- termini.
A conservative substitution is a substitution of one
amino acid for another of the same exchange group, the
exchange groups being defined as follows
I Gly, Pro, Ser, Ala (Cys) (and any nonbiogenic,
neutral~amino acid with a hydrophobicity not
exceeding that of the aforementioned a.a.'s)
II Arg, Lys, His (and any nonbiogenic, positively-
charged amino acids)
III Asp,, Glu, Asn, Gln (and any nonbiogenic
negatively-charged amino acids)
TV Leu, Ile, Met, Val (Cys) (and any nonbiogenic,
'aliphatic, neutral amino acid with a
hydrophobicity too high for I above)
V Phe, Trp, Tyr (and any nonbiogenic, aromatic
neutral amino acid with a hydrophobicity too high
f or I above ) .
Note that Cys belongs to both I and IV.
Residues Pro, Gly and Cys have special conformational
roles. Cys participates in formation of disulfide bonds.
Gly impart s flexibility to the chain. Pro imparts rigidity
to the chain and disrupts cx helices. These residues may be.
essential in certain regions of the polypeptide, but '
substitutable elsewhere.
One " two or three conservative substitutions are more
likely.to be tolerated than a larger number. ,
''"Semi-conservative substitutions" are defined herein as
being substitutions within supergroup I/II/III or within , .
supergroup IV/V, but not within a single one of groups I-V.,
They also include replacement of any other amino acid with
alanine.A If a substitution is not conservative, it
preferably is semi-conservative. ~ ' .
"Non-conservative substitutions" are substitutions
'35 which are not "conservative" or "semi-conservative".
"Highly conservative substitutions" are a subset of
conservative substitutions, and are exchanges of amino acids
.,within the groups Phe/Tyr/Trp, Met/Leu/Ile/Val,:His/Arg/Lys,
Asp/Glu and Ser/Thr/Ala.' They are more,likely to be
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tolerated than other conservative substitutions. Again, the
smaller the number of substitutions, the more likely they
are to be tolerated.
5 "Conservatively 'Identical"
A protein (peptide) is conservatively identical to a
reference protein (peptide) it differs from the latter, if
at all,. solely by conservative modifications, the protein
(peptide remaining at least seven amino acids long if the
10 reference protein (peptide) was at least seven amino acids
long .
A protein is at least semi-conservatively identical to
a reference protein (peptide) if it differs from the latter,
if at all, solely by semi-conservati''ve or conservative
15 modifications.
A protein (peptide) is nearly conservatively identical
to a reference protein (peptide) if it differs from the
latter, if at all, solely by one or more conservative
modifications and/or a single nonconservative substitution.
20 It is highly conservatively identical if it differs, if
at all, solely by highly conservative substitutions. Highly
conservatively identical proteins are preferred to those
merely conservatively identical. An absolutely identical
protein is even more preferred.
The core sequence of a reference protein (peptide) is
the largest single fragment which retains at least 100 of a
particular specific binding activity, if one is specified,
~30 or otherwise of, at,least one specific binding activity of
the referent: If the referent has more than one specific
binding activity, it may have more-than one core sequence,
,'
and these may overlap or not.
If it is taught that a peptide of the present invention
3.5 may have.a particular similarity relationship (e. g.,
markedly identical) to a reference protein (peptide),'
.p referred peptides are those which comprise a sequence
having that~relationship to a core sequence of the reference
protein (peptide), :but with, internal insertions or deletions
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56
in either sequence excluded. Even more preferred peptides
are those whose entire sequence has that relationship, with
the same exclusion , to a core sequence of that,reference
protein (peptide) .
Library
The term "library" generally refers to a collection of
chemical or biological entities which are related in origin,,
structure, and/or function, and which can be screened
simultaneously for a property of interest.
Libraries may be classified by how they are constructed
(natural vs. artificial diversity; combinatorial vs.
noncombinatorial), how they are screened (hybridization,
expression, display), or by the nature of the screened
library members (peptides, nucleic acids, etc.).'
In a "natural diversity" library, essentially all of
the diversity arose without human intervention. This would
be true, for example, of messenger RNA extracted from a non-
engineered cell.
In a "synthetic diversity" library, essentially all of
the diversity arose deliberately as a result of human
intervention. This would be'true for example of a
combinatorial library; note that a small level of natural
diversity could still arise as a result of spontaneous
mutation.. It would also be true of a noncombinatorial
library of compounds collected from diverse sources, even if
they -were' all natural products . ; ,
In a "non-natural diversity" library, at least some o'f
the diversity arose deliberately through human~intervention.
In'a "controlled origin" library, the source of the
diversity is limited in some way. A limitation might, be to
cells of a particular individual, to a particular species,
or to.~a particular genus, or, more complexly to individuals
3'5 of a particular species who are of a particular age, sex,
physical~condition,.geographical location,,occupation and/or;
familial relationship. ,Alternatively or,additionally, its,
~,
might 'be to cells of a particular tissue or. organ., Or ,it
w could be cells exposed to partiCUlar pharmacological,
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environmen.~al, or pathogenic conditions. Or the library
could be o f chemicals, or a particular class of chemicals,
produced by such cells.
In a "controlled structure" library, the library
members are deliberately limited by the production
conditions. to particular chemical structures. For example,
if they arm oligomers, they may be limited in length and
monomer composition, e.g. hexapeptides composed of the
twenty genetically encoded~amino acids.
Hybridizat Son Library
In a hybridization library, the library members are
nucleic ac ids, and are screened using a nucleic acid
hybridizat ion probe. Bound nucleic acids may then be
amplified, cloned, and/or sequenced.'
Expression Library ,
In an expression library, the screened library members
are gene expression products, but one may also speak of an
underlying library of genes encoding those products. The
library is made by subcloning DNA encoding the library
members (or portions thereof) into expression vectors (or
into cloning vectors which subsequently are used to
construct expression vectors), each vector comprising an
2.5 expressible gene encoding a particular library member,
introducing .the expression vectors into suitable cells, and
expressing the genes so the expression products are
produced. ' .
In one embodiment, the expression products are ,
secreted, s o the library can be screened using an affinity ,
reagent, such as an antibody~or receptor. The bound
expression products may be sequenced,directly, or their ,~
sequences inferred by, e.g., sequ.encing at least the
variable portion of the encoding DNA.
In a s econd.embodiment, the cells are lysed,.thereby',
exposing the expression products, and,the latter are
screened wi. th the of f inity reagent . ~ v ' '-
In a third embodiment, the cells;express the' library
members in such a manner'that they are~.displayed on.the
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surface of the cells, or on the surface of viral particles
produced by the cells. (See. display libraries, below).
In a fourth embodiment, the screening is not for the
ability of the expression product to bind to an affinity
reagent, but rather for its ability to alter the phenotype
of the host cell in a particular detectable manner. Here,
the screened library members are transformed cells, but
there is a first underlying library of expression products
which mediate the behavior of~the cells, and a second
underlying library of genes which encode those products.
Display Library
In a display library, the library members are each
conjugated to, and displayed upon, a support of some kind.
The support may be living (a cell or virus), or nonliving
(e.g. , a bead or plate) .
If the support is a cell or virus, display will
normally be effectuated by expressing a fusion protein which
comprises the library member, a carrier moiety allowing
integrat i on of the fusion protein into the surface of the
cell or virus, and optionally a lining moiety. In a
variation on this theme, the cell coexpresses a first fusion
comprising the library member and a linking moiety L1, and a
second fusion comprising a linking moiety L2 and the carrier
moiety. L1 and L2 interact to associate the first fusion
with the second fusion and hence,,indirectly, the library .
member with the surface of the cell or virus.
,.
Soluble L ibrary
In a soluble.library, the library members are free in .
solution. A.soluble library may be produced directly, or
one may f irst'make a display library and then release the
library members from their supports . '.
Encapsulated Library. '
In an encapsulated library, the library members are '
inside ce 11s or liposomes. Generally speaking, encapsulated
libraries are used to 'store the library members for vfutilre ,'
use; the members are extracted in some way for screening ~ "
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purposes. However, if they differentially affect the
phenotype of the cells, they may be screened indirectly by
screening the cells.
cDNA Librarv
A cDNA library is usually prepared by extracting'RNA
from cells of particular origin, fractionating the RNA to
isolate the messenger RNA (mRNA has a poly(A) tail, so this
is usually done by oligo-dT affinity chromatography),
synthesizing complementary DNA (cDNA) using reverse
transcriptase, DNA polymerase, and other enzymes, subcloning
the cDNA into vectors, and introducing the vectors into
cells. Often, only~mRNAs or cDNAs of particular sizes will
be used, to make it more likely that the cDNA encodes a
functional polypeptide.
A cDNA library explores the natural diversity of the
transcribed DNAs of cells from a particular source. It is
not a combinatorial library.
A cDNA lsbrary may be used to make a hybridization
library, or it may be used as. an (or to make) expression
library.'
Genomic DNA Librarv
A genomic DNA',library is made by extracting DNA from a
particular source, fragmenting the DNA, isolating fragments
of a particular size range, subcloning the DNA fragments
into vectors, and introducing the vectors into cells.
Like a cDNA library, a genomic DNA library is a natural ,
diversity library, and not a combinatorial library. A '
genomic DNA library may be used the same way as a~,cDNA
library. ~ ,
Synthetic DNA library , ,
A synthe tic DNA library may be screened directly (as a
hybridization library), or used in the creation of',an '
expression. or display library of peptides/proteins.
Combinatorial Libraries
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The term "combinatorial library" refers to a library in
which the indivi dual members are either systematic or random
combinations of a limited set of basic elements, the
properties of each member being dependent on the choice and
5 location of the elements incorporated into it. Typically,
the members of t he library are at least capable. of being
screened simultaneously. Randomization may be complete or
partial; some positions may be randomized and others
predetermined, and at random positions, thevchoices may be
10 limited in a pre determined manner. The members of a
combinatorial library. may be oligomers or polymers, of some
kind, in which the variation occurs through the choice of
monomeric building block at one or more positions of the
oligomer or polymer, and possibly in terms of the connecting
15 linkage,~or the 1 ength of the oligomer or polymer, too. Or
the members may be nonoligomeric molecules with a standard
core structure, 1 ike the 1,4-benzodiazepine structure, with
i
the variation being introduced by'the choice of substituents
at particular variable sites on the core structure. Or the ,
20 members may be nonoligomeric molecules assembled like a
jigsaw puzzle, but wherein each piece has both one or more
variable moieties (contributing to library diversity) and
one or more const ant moieties (providing the functionalities
for coupling the piece in question to other pieces).
25 Thus, in a typical combinatorial library, chemical
building blocks are, at least partially randomly combined
into a large number (as high as 1015) of different compounds,
which are then simultaneously screened for binding (or
other) activity against one or more targets. ,
30 In a "simple combinatorial library";.all of,the members .
belong to the same class of compounds (e. g., peptides) and'
can be synthesized simultaneously. .A "composite '
combinatorial library" is a mixture of two or more simple
,libraries, e.g., DNAs and peptides,, or peptides,, peptoids,
35 and PNAs, or bent odiazepines and carbamates., The number of
component simple libraries in a composite library will, of
course, normally be smaller than the average numbeW of v
members in each s imple library, a~sv otherwise' the'. advantage
of a library'over individuah,synthesis is small.
. , . . , , ~1 ,
;, '; ~ ,
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61
Libraries of thousands, even millions, of random
oligopeptides have been prepared by chemical synthesis
(Houghten. et a1. , Nature, 354 : 84-6 (1991) ) , or gene
expression (Marks et: al., J Mol Biol, 222:581-97(1991)),
displayed on chromatographic supports (Lam et al., Nature,
354:82-4(1991)), ins ide bacterial cells (Colas et al.,
Nature, 380:548-550(1996)), on bacterial pili (Lu,
Bio/TeChnology, 13:3 66-372(1990)), or phage (Smith, Science,
228:1315-7(1985)), and screened for binding to a variety of
targets including antibodies (Valadon et al., J Mol Biol,
261:11-22(1996)), cellular proteins (Schmitz et al., J Mol
Biol, 260:664-677(19 96)), viral proteins (Hong and
Boulanger, Embo~J, 14:4714-4727(1995)), bacterial proteins
(Jacobsson.and~Frykberg, Biotechniques, 18:878-885(1995)),
nucleic acids (Cheng et al., Gene, 1,71:1-8(1996)), and
plastic (Siam et a1. , J Chem Inf Comput Sci, 34 : 588-
593 (1994) ) .
Libraries of proteins (Ladner, USP 4,664,989), peptoids
1(Simon et al., Proo Natl Acad Sci U S A, 89:9367-71(1992)),
nucleic acids (Ellington and Szostak, Nature,
246:818(1990)), carbohydrates, and small organic molecules
(Eichler et al., Med Res Rev, 15:481-96(1995)) have also
been prepared or suggested for drug screening purposes.
The first combinatorial libraries were composed of
peptides or proteins, in which all or selected amino acid
positions were randomized. Peptides and~proteins can exhibit
high and specific binding activity, arid can act.as
catalysts. In consequence, they,are of great importance in
' biological systems.
Nucleic acids have also been used in Combinatorial
libraries. Their great advantage,is the ease with which a.
nucleic acid with appropriate binding activity can be
amplified. As a result, combinatorial libraries composed of
nucleic acids can be of.low redundancy and hence, of high
. 35 diversity.' ~ , '
There has. also been much interest iri~combinatorial'
libraries'based on small molecules, which,are more suited to
. , '
pharmaceutical use, especially those which, like
benzodiazepines,~ belong to a chemical class which,has , ,
i , . , ..
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already yielded useful pharmacological agents. The
techniques of combinatorial chemistry have been recognized.
as the most efficient means for finding small molecules that
act on these targets. At present, small molecule
combinatorial chemistry involves the synthesis of either
pooled or discrete molecul as that present varying arrays of
functionality on a common scaffold. These 'compounds are'
grouped in libraries that are then screened against the
target of interest either for binding or for inhibition of
biological activity.
The size of a library is the number of molecules in it.
~'he simple diversity of a 1 ibrary is the number of unique
structures in it. There is no formal minimum or maximum
diversity. If the library has a very low diversity, the
1 ibrary has little advantage over just synthesizing and
screening the members individually. If the library is of
very high diversity, it may- be inconvenient to handle, at
1 east without automatizing the process. The simple
diversity of a library is preferably at least 10; 10E2,
_ 20 30E3, 10E4, 10E6, 10E7, 10E 8 or 10E9, the higher the better
under most circumstances. Z'he simple diversity is usually
not more than.10E15, and more usually not more than 10E10.
The average sampling 1 evel is the size divided by the
simple diversity. The expected average sampling level must
be high enough to provide a reasonable assurance'.that,"if a
given structure were expect ed, as a consequence of the
1 ibrary design, to be present, that'the actual average
sampling level will be' high enough so that the.structure, if
satisfying the screening criteria, will yield a positive
result whm the library is screened. Thus, the preferred
' average sampling level is a function of the detection limit,
cwhich in ; turn is a function of the strength of the signal .to
be screened. . v
There are more complex measures of diversity~than ,
~s imple diversity. These at tempt to take into account the
degree of structural difference between the various unique
sequences. These more compl ex'measures are usually usedin
t he context of small. organi c compound libraries, see below.
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The library members may be presented as solutes in
solution, or immobilized on some form of support. In the
latter case, the support may be living (cell, virus) or
nonliving (bead, plate, etc_). The supports may be separable
(cells, virus particles, beads) so that binding and
nonbinding members can be separated, or nonseparable
(plate). In the latter case, the members will normally be
placed on addressable positions on the support. The
advantage of a soluble library, is that there'is.no carrier
moiety that could interfere with the binding of the members
to the support. The advantage of an immobilized library is
that it is easier to identify the structure of the members
which were positive.
When screening a solubl a library, or one with a
separable support, the target is usually immobilized. When
screening a library on a nonseparable support, the target
will usually be labeled.
Oligonucleotide Libraries
An oligonucleotide library is a combinatorial library,
at least some of whose members are single-stranded
oligonucleotides having thre a or more nucleotides connected
by phosphodiester or analogous bonds. The ol,igonucleotides
may be linear, cyclic or branched, and may include non-
nucleic acid moieties. The, nucleotides are not limited to
the nucleotides normally found in DNA or RNA. For examples
of nucleotides modified to increase nuclease resistance and
chemical~stability of aptame rs, see Chart l in Osborne and
Ellington, Chem. Rev. ,. 97 : 349-70, (1997) . For screening of ,
RNA, see Ellington and Szostak, Nature, 346:.818-22 (1990).
There is no formal minimum or maximum ~siz~e for these ~~
oligoriucleotides. However, th.e number of conformations which
an oligonucleotide can assume increases exponentially with.
its length in bases. Hence, a longer oligonucleotide~.is
more likely to be able to fold to adapt' itself to a protein
surface: On the other hand, while very'long~molecules can
be;synthesized and screened, unless they provide a much .
superior affinity to that of shorter molecules, they~are not
likely to be found in the selected 'population; for ,the
,.
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reasons explained by Osborne and Ellington (1997). Hence,
the libraries of the present invention are preferably
composed of oligonucleotides having a length of 3 to 100
bases, more preferably 15 to 35 bases. The oligonucleotides
in a given library may be of the same or of different
lengths.
Oligonucleotide libraries have the advantage that
libraries of very high diversity (e.g. , 1015) are feasible,
and binding molecules are readily amplified in vitro by
polymerase chain reaction (PCR). Moreover, nucleic acid
molecules can,have very high specificity and affinity to
targets.
In a preferred embodiment, this invention prepares and
screens oligonucleotide libraries by the SELEX method, as
described in King and Famulok, Molec. Biol. Repts., 20: 97-
107 (1994); L. Gold, C. Tuerk. Methods of producing nucleic
acid ligands, US#5595877; Oliphant et al. Gene 44:177 '
(1986) .
The term "aptamer" is conferred on those
oligonucleotides which bind the target protein. Such
aptamers may be used to. characterize the~target protein,
both directly (through identification of the aptamer and the
points of contact between the aptamer and the protein) and
indirectly (by use of the aptamer as a ligand to modify the
~25 chemical reactivity of the protein). '
In a classic oligonucl otide, each nucleotide (monomeric
. unit) is.composed of a phosphate group, a sugar moiety, and
either a purine or a pyrimi dine base. In DNA, the sugar is
deoxyribose arid in RNA it i s ribose. The nucleotides are
linked by 5'-3' phosphodies ter bonds.
The~deoxyribose phosphate backbone of DNA can be
modified to increase resist ante to~nuclease and to increase
penetration of cell membranes. Derivatives such as mono- or
dithiophosphates, methyl phosphonates, boranophosphates, ,
formacetals, carbamates, siloxanes,, and dimethylenethio- -
sulfoxideo- and-sulfono -linked species are known in the'
art . . . ' '
-.
Peptide Library '~ '
~ ,
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A peptide is composed of a plurality of amino acid
residues joined together by peptidyl (-NHCO-) bonds. A .
biogenic peptide is a peptide in which the residues.are all
genetically encoded amino acid residues; it is not necessary
5 that the biogenic peptide actually be produced by gene
expression.
Amino acids are the basic building blocks with which
peptides and proteins are constructed. Amino acids possess
both an amino group (-NHZ) and a carboxylic acid group (-
10 COOH). Many amino acids, but not all, have the alpha amino
acid structure NHz-CHR- COOH, where R is hydrogen, or any of a
variety of functional groups.
Twenty amino acids are genetically encoded: Alanine,
Arginine, Asparagine, .Aspartic Acid, Cysteine, Glutamic.
15 Acid, Glutamine, Glycine, Histidine, Isoleucine,~Leucine,
Lysine, Methionine, Phenylalanine, Proline, Serine,
Threonine, Tryptophan, Tyrosine, and Valine. Of these, all
save Glycine are optically,isomeric, however,lonly the L-
' form is found in humans. Nevertheless, the D-forms of these
20 amino acids do have biological significance; D-Phe, for
example, is a known analgesic. '
Many,other amino acids are also known,'including: 2-
Aminoadipic acid; 3-Amz.noadipic acid; beta-Aminopropionic
acid; 2-Aminobutyric ac id; 4-Aminobutyric acid (Piperidinic
25 acid);6-Aminocaproic ac id; 2-Aminoheptanoic acid; 2-
Aminoisobutyric acid, 3-Aminoisobutyric acid; 2-Aminopimelic
acid; 2,4-Diaminobutyri.c acid; Desmosine; 2,2'-
Diaminopimelic acid;.2,3-Diaminopropionic acid; N-
Ethylglycine; N-Ethylasparagine; Hydroxylysine;, allo-
30. Hydroxylysine; 3-Hydroxyproline; 4-Hydroxyproline;~ .'
Isodesmosine; allo-Isol eucine; N-Methylglycine~(Sarcosine);
N-Methylisoleucine; N-Methylvaline;~ Norvaline; Norleucine;
' and Ornithine.
Peptides are constructed by condensation of amino acids
35 and/or smaller peptides. The amino group of one amino acid .w
(or peptide) reacts,wit h the carboxylic acid group of a.
second amino acid. (or pepode) to form a peptide . (-NHCO-)
bond, ;releasing- one molecule of ~~water.~ Therefore, when an
'' amino acid is incorporated into'a peptide,'-it should;
!
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technically speaking. be referred to as an amino acid .
residue. The core of that residue is the moiety which
excludes the -NH and -CO linking functionalities which
connect it to other residues: This moiety consists of one
or more main chain atoms (see below) and the attached side
chains.
The main chain moiety of each amino acid consists of
the -NH and -CO linking functionalities and a core main
chain moiety.. Usually, the latter is a single carbon atom.
However, the core main chain moiety may include additional
carbon atoms, and may al so include nitrogen, oxygen or
sulfur atoms, which together form a single chain. In a ,
preferred embodiment, tha core main chain atoms consist
solely of carbon atoms.
The side chains are attached to the core main chain
atoms. For alpha amino ac ids, in which the side chain is
attached to the alpha carbon, the C-1, C-2 and N-2 of each
residue form the repeating unit of the main chain, and the
word "side chain" refers to, the C-3 and higher numbered
carbon atoms and their substitu'ents. It also includes H
atoms attached to the main chain~atoms.
Amino acids may be classified according to the number
of carbon atoms which appear in the main chain between the
carbonyl carbon and amino nitrogen atoms which'participate
in the peptide bonds. Among the 150 or so amino acids which
occur in nature, alpha, beta, gamma and delta amino acids
are known. These have 1 -4 intermediary carbons. Only alpha
amino acids occur in proteins. Proline is a special case of
an alpha amino acid; its side chain also binds to the
peptide bond nitrogen...
For beta and higher order amino acids, there is a'.
' choice as to which main chain core carbon aside chain other '
than H is attached to.~ The~preferred attachment site is the
C-2 (alpha) carbon, i.e., the one adjacent to. the carboxyl
carbon of the -CO linking functionality. It is also possible
' for more than one main chain atom to carry~a'side chain
other than H. However, in a preferred. embodiment,. only one',
main chain core atom carries a side chain ,other than H. ' . ~~
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A main chain carbon atom may carry either one or two
side chains; one is more common. A side chain may be
att ached to a main chain carbon atom by a single or a double
bored; the former is more common.
A simple combinatorial peptide library is one whose
members are peptides having thre a or more amino acids
connected via peptide bonds.
The peptides may be linear, branched, or cyclic, and
may covalently or noncovalently include nonpeptidyl
.moieties. ,The amino acids are not limited to the naturally
occurring or to the genetically encoded amino acids.
A biased peptide library, is one in which one or more
(but not all) residues. of the peptides are constant
res~..dues .
Cyclic Peptides
Many naturally occurring peptides are cyclic.
CycZ.ization is a common mechanism for stabilization of
peptide conformation thereby ache eying improved association
of the peptide with its ligand and hence improved biological
activity. Cyclization is usually achieved by intra-chain
cyst ine formation, by formation o f peptide bond between side
chains or between N- and C- termi pals. Cyclization was
usually achieved by peptides in solution, but several
pubL ications.have appeared that describe cyclization of
pept ides on beads.
~. A peptide library may be an oligopeptide library or a
protein library. , w
Oligopeptides
Preferably, the oligopeptide s are at least five, six,
seven or eight amino acids in length. ~ Preferably, they are
composed of less than 50, more preferably less than 20 amino
acids .
In the' case of an oligopeptide , library, ~ all or just
some ,of the' residues maybe variable. The oligopeptide may
be unconstrained, ,or constrained to a. particular
',conformation by, e'.g. , the partics.pation of constant
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cysteine residues in the format son of a constraining
disulfide bond.
Proteins
Proteins, like oligopeptide s, are composed of a
plurality of amino acids, but the term protein is usually
reserved for longer peptides, which are able to~fold into a
stable conformation. A protein may be composed of two or
more polypeptide chains, held together by covalent or'
' noncovalent crosslinks. These may occur in a homooligomeric
or a heterooligomeric state. .
A peptide is considered a protein if it (1) is at least
50 amino acids long, or (2) has at least two stabilizing
covalent crosslinks (e. g., disulfide bonds). Thus,
conotoxins are considered proteins.
Usually, the proteins of a protein library will be
characterizable as having both constant residues (the same
for all proteins in the library) and variable residues
(which vary from member to member). This is simply because,
for a given range of va.riation.at each position, the'
'sequence space (simple diversity) grows exponentially with
the~number of residue positions, so at some point it becomes
inconvenient. for all residues of a peptide to be variable
positions. Since proteins are usually larger than
oligopeptides, it is morelcommon for protein libraries than
oligopeptide libraries to feature variable positions..
In the case of a protein library, it is desirable to ,
focus the mutations at those sit es which are tolerant. of
mutation...,These may, be determined by alanine scanning
mutagenesis or by comparison of the protein.s'equence to that
of~homologous proteins'of,similar activity. It is also more
;,likely that mutation.,of surface residues will directly
affect binding., Surface residue s may be determined by
inspecting a 3D structure of the protein, or by labeling the
~ surface and then ascertaining Wh1 Ch residues, have received
'labels: They may also be inferred by identifying regions of
high hydrophilicity':~,vithin the protein. . '
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Because proteins are often alt eyed at some sites but
not others, protein libraries can be considered a special
case of the biased peptide library_
There are several reasons that one might screen a
protein library instead of an oligopeptide library,
including (1) a particular protein, mutated in the library,
has the desired activity to some degree already, and (2)~the
oligopeptides are not expected to have a sufficiently high
affinity or specificity. since they do not have a stable
conformation.
When the protein library is based on a parental protein
which does not have the desired activity, the parental
protein will usually be one which i s of high stability
(melting point >= 50 deg. C.) and/o r possessed of
hypervariable regions.
The variable domains of 'an ant ibody possess
hypervariable regions and hence, in some embodiments, the
protein library comprises members which comprise a.mutant of
VH or VL chain, or a mutant ,of an antigen-specific binding
fragment of such a chain. VH and VL chains are usually each
about 110 amino acid residues, and are held in proximity by
a disulfide bond between the adjoing CL.and CH1 regions to
form a variable domain. Together, th.e VH, VL, CL and CHl
form an Fab fragment.
In human heavy,chains, the hypervariable regions are at
31-35, 49-65, 98-111 and 84-88, but ,only the first three are
involved in antigen binding. There is variation among VH
and VL chains at residues outside the~hypervariable regions,
but to a much lesser degree. , '
,30 A sequence is considered a mutant of a VH or VL chain
if it is at least 80% identical to ~a naturally occurring VH
' or VL chain at all residues outside the hypervariable~
region. '
In a preferred embodiment, such. antibody library
members comprise both at least one VH chain and at least one
VL chain, at least one of which is a mutant chain, and which .
chains may be derived from the same or different, antibodies.
The VH and VL chains may be covalei~.tly joined. by a suitablel~',,
linker moiety, as in a "single chain antibody",'.or they'may
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be noncovalently joined, as in a natural 1y occurring
variable domain..
If the joining is noncovalent, and the library is
displayed on cells or virus, then either the VH or the VL
5 chain may be fused to the carrier surfac e/coat protein. The
complementary chain may be co-expressed, or added
exogenously to the library.
The members may further comprise some or all of an
antibody constant heavy and/or constant light chain, or a
10 mutant thereof.
Peptoid Library , '
A peptoid is an analogue of a peptide in which one or
more of the peptide bonds ,(=NH-CO-) are replaced by
' 15 ' pseudopeptide bonds, which may be the same or different. It
is not necessary that all of the peptide bonds be replaced,
i.e., a peptoid may include one or more conventional amino
acid residues, e.g., proline.
A peptide bond has two small divalent linker elements,
20 -NH- and -CO-. Thus, a preferred class of psuedopeptide
bonds are those which consist of two sma 11 divalent linker
elements. Each may be chosen independent 1y from the group
consisting of amine (-NH-), substituted amine (-NR-),
carbonyl (-CO-) , thiocarbonyl ~ (-CS-) ,methylene (-CH2-) ,
25 monosubstituted methylene (-CHR-), disubstituted methylene
(-CR1R2-), ether (-0-) and thioether (-S-). The more
preferred pseudopeptide bonds include:
N-modified -NRCO-
Carba ~ -CHZ-CH2-
30 Depsi ~ -CO-O-
' Hydroxyethylene ~ -CHOH-CH2-
Ketomethylene ~ -CO-CHz-
Methylene-Oxy -CH2-O-
Reduced -CHI-NH-
y 35 Thiomethylene -CHI-,S-
' Thiopeptide -CS-NH-
~, Retro-Inverso -CO-NH- '
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A single peptoid molecule may include more than one
kind of.pseudopeptide bond.
For the purposes of introducing diversity into a
peptoid library, one may vary (1) the side chains attached
to the core main chain atoms of the monomers 1 inked by the
pseudopeptide bonds, and/or (2) the side chains (e.g., the -
R of an -NRCO-) of the pseudopeptide bonds. Thus, in one
embodiment, the monomeric units which are not amino acid
residues are of the structure -NR1-CR2-CO-, where at leas
one. of R1 and R2 are not hydrogen. If there s s variability
in the pseudopeptide bond, this is most conveniently done by
using an -NRCO- or other pseudopeptide bond with an R group,
and varying the R group. In this event, the R group will
usually be any of the side chains characteriz~.ng the amino
' acids of peptides, as previously discussed.
If the R group of the pseudopeptide bond is not
variable, it will usually be small, e.g., not more than 10
atoms (e. g., hydroxyl, amino,,carboxyl, methyl, ethyl,
propyl ) ,
If the conjugation chemistries are compatible, a simple
combinatorial library may include both peptide's and
peptoids.
Peptide Nucleic Acid Library
A PNA oligomer is here defined as one,cornprising a
plurality of units,~at least one of which is a PNA monomer
which comprises a side chain comprising a nucl eobase. For
nucleobas,es,~see USP 6,077,835.
The classic PNA oligomer is composed of <2-
3.0 aminoethyl)glycine units, with nucleobases attached by , w, ,
methylene carbonyl linkers. That is, it has. the structure
H- (-HN-CH2-CHZ-N(-CO-CHZ-B)-CH2-CO-)n -OH
35v where the outer parenthesized substructure is the PNA
monomer.;
In this structure, the' nucleobase B is separated ~ from ., ' '
the':backbone N by three bonds, and the points of attachment ~,.
,;
', . .
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of the side chains are separated by six bonds. The
nucleobase may be any ofthe bases included in the
nucleotides discussed in connection with oligonuc leotide
libraries. The bases of nucleotides A, G, T, C and U are
preferred.
A PNA oligomer may further comprise one or mire amino
acid residues, especially glycine and proline.
One can readily envision related molecules i n which (1)
the -COCH2- linker is replaced by another linker, especially
one composed of two small divalent linkers as def fined
previously,, (2) a side chain is attached to one of the three
main~chain carbons not participating in the pepti de bond
(either instead or in addition to the side chain attached to
the N of the classic PNA).; and/or (3) the peptide bonds~are
replaced by pseudopeptide bonds as disclosed previously in
the context of peptoids. '
PNA oligomer libraries have been made; see e.g. Cook,
6,204,326.
Small Organic Compound Library
The small organic compound library ("compound library",
for short) is a combinatorial library whose members are
suitable for use as drugs if, indeed, they have t he ability
to'nlediate a biological activity of the target protein.
' Peptides have certain disadvantages as drugs. These
include susceptibility to.degradation by serum proteases,
and difficulty in penetrating cell membranes. Preferably,
' all or most of the compounds of the compound library avoid,
or at'least do not suffer to the same degree, one or more of
the pharmaceutical~disadvantages of peptides.
' - ~ In designing a compound library, it.is helpf~:ul to bear
in mind the methods; of molecular modification typically used
to'obtain,new drugs. Three basic kinds of modification. may
be identified: disjunction, in which a lead drug is ,
simplified to identify its component, pharmacophoric,.~ '
moieties; con-iunction, in which two or more knowri ,
pharmacoph'oric moieties, which maybe the same or different,
are associated, 'covalexitly .or noncovalently, to. form a new
drug; and alteration, in'~ which one moiety 'is, replaced by
. , ,
;..
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another which may be similar or different, but which i ~ not
in effect a disjunction or conjunction. The use of the
terms "disjunction", "conjunction" and "alteration" is
- intended only to connote the structural relationship of the
end product to the original leads, and not how the new drugs
are actually synthesized, although it is possible that the
two are the same.
The process of disjunction is illustrated by the
evolution of neostigmine (1931) and edrophonium (1952) from
physostigmine (1925). Subsequent conjunction is illustrated
by demecarium (1956) and ambenonium (1956).
Alterations may modify the size, polarity, or electron
distribution of an original moiety. Alterations include
ring closing or opening, formation of lower or higher
homologues, introduction or saturation of double bonds
introduction of optically active centers, introduction,
removal or replacement of bulky groups, isosteric or
bioisosteric substitution, changes in the position or
orientation of a group, introduction of alkylating groups,
and introduction, removal or replacement of groups with- a'~
view toward inhibiting or promoting inductive
(electrostatic) or. conjugative (resonance) effects.
Thus, the substituents may include,electron accept ors
and/or electron donors. Typical electron donors (+I)
include -CH3, -CHzR, -CHR2, -CR3 and -COO'. Typical electron
acceptors (-I) include -NH3+, , -NR3+, -N02, -CN, -COOH, - COOR,
-CHO, -COR, -COR, -F, -C1, -Br, -OH, -OR, -SH, -SR, =CH=CHz,'
-CR=CRS, and -C=CH. '
The substituents may also include those which increase
or decrease.electronic density in conjugated systems: The
former (+R)' groups include -CH3; -CR3,v-F, -C1, -Br, -Ir, -OH,
-OR, -OCOR, -SH, -SR, -NH2, -NR2, and -NHCOR. The later (-R)
groups include -N02, -CN, , -CHC, -COR, ~ -COOH, -COOK, ~-COLVH2, .
-SOzR and -CF3.
Synthetically speaking, the modifications may be
achieved by a variety of unit processes, including
nucleophilic and electrophilic substitution, reduction~.and
oxidation, addition elimiriatlom,~double~bond cleavage, and
cyclization.
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For the purpose of constructing a library, a compound,
or a family of compounds, having one or more pharmacological
w activities (which need not be relatred to the known or
suspected activities of the target protein), may be
disjoined into two or more known o~ potential pharmacophoric
moieties. Analogues of each of the se moieties may be
identified, and mixtures of these analogues reacted so as to
reassemble compounds which have sotr~.e similarity to the
original.lead compound. It is not necessary that all
members of the library possess mole ties analogous to all of
the moieties of the lead compound.
The design of a library may be illustrated by the
example of the benzodiazepines. Several benzodiazepine
drugs., including~chlordiazepoxide, diazepam and oxazepam,
', have been used as anti-anxiety drug s. Derivatives of
benzodiazepines have widespread bio logical activities;
derivatives have been reported to act not only as
anxiolytics, but also as anticonvul pants; cholecystokinin
(CCK) receptor subtype A or B,~kapp a opioid receptor,
platelet activating factor, and HIV transactivator Tat
antagonists, and GPIIbIIa, reverse ~ranscriptase and ras
farnesyltransferase inhibitors.
The benzodiazepine structure has been disjoined into a
2-aminobenzophenone, an amino acid, and an alkylating agent..
~~25 See Bunin, et al., Proc. Nat. Acad. Sci. USA, 91:4708
(1994). Since only a few 2-aminober~.zophenone derivatives
are commercially available, it was S ater disjoined into 2-
aminoarylstannane, an acid,chloride' an amino acid, andlanl '
alkylating agent. Bunin, et al., Meth. Enzymol., 267:448
~ ~ (1996) . The arylstannane may be considered the core
' structure upon which the other moietrie's are substituted, for 1'
all four may be considered equals which are conjoined.to
~, make each library member.
A basic library synthesis plan and member structure is
shown in Figured of Fowlkes, et a1_, U.S. Serial No.
,08/740,671, incorporated by reference in its entirety. The
-,acid,chloride~building block introduces variability at the Rte,
site. The R2 site is introduced by the amino acid, and'~the1
R3~, site , by the, alkylating agent . Th a R4 site 'is inherent in ~,
' ~ ,
. , ,
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the arylstannane. Bunin, et al. generated a 1, 4-
benzodiazepine library of 11,200 different derivatives
prepared from 20 acid chlorides, 35 amino acids, and 16
alkylating agents. (No diversity was introduced at R4; this
5 group was used to couple the molecule to a solid phase.)
According to the Available Chemicals Directory (HDL
Information Systems, San Leandro CA),, over 300 acid
chlorides, 80 Fmoc-protected amino acids and 800 alkylating
agents were available for purchase (and more, of course,
10 could be synthesized). The particular moieties used were
chosen to maximize structural dispersion, while ~.imiting the
numbers to those conveniently synthesized in the wells of a
microtiter plate. In choosing between structurally similar
compounds, preference was given to the least substituted
15 compound.
The variable elements included both aliphatic and
aromatic groups. Among the aliphatic groups, both acyclic
and cyclic (mono- or poly-) structures, substituted or not,
were tested.' (While all of the acyclic groups were linear,
20 it would have been feasible to introduce a branched
aliphatic). The aromatic groups featured either single and.
multiple rings, fused or not, substituted or not, and with
heteroatoms or not. The secondary substitutents included -
NH2, -OH, -OMe, -CN, -C1,,-F, and -COOH. While not used,
25 spacer moieties, such as -O-, -S-, -00-, -CS-, -NH-, and -
NR-, could have been incorporated:
Bunin et al. suggest that instead of using a 1, 4-
benzodiazepine as a core structure, one may,instead use a 1,
4-benzodiazepine-2, 5-dione structure.
30 As noted by Bunin et al., it is advantageous, although
not necessary, to use a linkage strategy which,leaves no
trace of; the linking furictionality,,as this permits
construction of a more diverse. library.
Other combinatorial nonoligomeric compound libraries
35 known or,suggested in the~art have been based on,:carbamates,
mercaptoacylated pyrroli,dines, phenolic agents, aminimides,
.N-acylamino ethers~(made from amino alcohols, aromatic.
' , hydroxy,acids,'l.and carboxylic acids), N-alkylamino ethers
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(made from aromatic hydroxy acids, amino alcohols and
aldehydes) 1, 4-piperazines, and 1, 4-piperazine-6-ones.
DeWitt, et al., Proc. Nat. Acad. Sc~.. (USA), 90:6909-13,
(1993) describe the simultaneous but separate, synthesis of
40 discrete hydantoins and 40 discrete benzodiazepines.
They carry out their synthesis on a solid support (inside a
gas dispersion tube), in an array format as opposed to ,
other conventional simultaneous synthesis techniques (e. g.,
in a well, or on a pin): The hydantoins were synthesized by
first simultaneously deprotecting and then treating each of ,
five amino acid resins with each of eight isocyanates. The
benzodiazepines were synthesized by trea-~ ing each of five
deprotected amino acid resins with each of eight 2-amino
benzophenone imines.
Chen, et al., J. Am. Chem. Soc., 116:2661-62 (1994)
described the .preparation of a pilot (9 member)
combinatorial library of formate esters. A polymer bead-
bound aldehyde preparation was "split" into three aliquots,
each reacted with one of three different ylide reagents.
The reaction products were combined, and then divided into
three new aliquots, each of which was reacted with a
different Michael donor. Compound identity was found to be
determinable on a single bead basis by gas
chromatography/mass spectroscopy analysis.
Holmes, USP 5,549,974 (1996) sets forth methodologies'
for the combinatorial,synthesis of libraries of
thiazolidinones and metathiazanones. These libraries are a
made by combination of amines, carbonyl compounds, and
thiols under cyelization conditions.
Ellman, USP 5,545,568 (1996) describes combinatorial
synthesis of benzodiazepines, prostaglanc~ins, beta-turn
mimetics, and glycerol-based compounds.. See'also Ellman,
USP 5,288,514.. ,
Summerton, tTSP 5, 506, 337 (1996) discloses methods of
preparing'a combinatorial library formed predominantly.of' ~,
rnorpholino subunit structures. , ' . .
Heterocylic combinatorial libraries are, reviewed
. ; ,
generally in Nefz~i, '~et~ al . , Chem. ' Rev. , 97:449-472 (1997) .' . ;
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For pharmacological classes, see, e.g., Goth, Medical '
Pharmacology: Principles and Concepts (C.V. Mosb~r Co.: 8th
ed. 1976); Korolkovas and Burckhalter, Essentials of
Medicinal Chemistry (John Wiley & Sons, Inc.: 19~ 6). For
synthetic methods, see, e.g., Warren, Organic Synthesis: The
Disconnection Approach (John Wiley & Sons, Ltd.: 1982);
Fuson, Reactions of Organic Compounds (John Wiley & Sons:
1966); Payne and Payne, How to do an Organic Syn~hesis
(Allyn.and Bacon, Inc.: 1969); Greene,. Protective Groups in~
.10 Organic Synthesis (Whey-Interscience) . For selection of
substituents, see e.g., Hansch and Leo, Substituant
Constants for Correlation Analysis in Chemistry and Biolo~y
(John Wiley & Sons: 1979). .
The library is preferably synthesized so that the
individual members remain: identifiable so that, L f a' member
is shown to be active, it is not necessary to an~.lyze it.
Several methods of identification have been proposed,
including:
(1) encoding, i.e., the attachment to each member of
ari identifier moiety which is more reacLily .
identified than the member proper. Thi s has the
disadvantage that the tag may itself influence the
activity of the conjugate.
(2) spatial addressing, e.g., each member i s
' 25 synthesized only at a particular coordinate on~or
in a matrix, or in a particular chamber. This
might be, for example, the location of a
. , ~ particular pin, or a particular well on a I '
microtiter plate, or inside a "tea bag" .
~ The present invention is not limited.to any particular form
' of identification. ~' '
However, it i's possible to simply characteri2e those
members of the library which are found to be acti~re, based ,
on. the characteristic spectroscopic indicia of the various
.building blocks.
Solid phase synthesis permits greater control over
which,derivatives are formed. However,, the' solid phase
could interfere with activity. To overcome this problem,'. ' '
~,, '~ ' ,.
', '
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some or all of the molecules of each member could be
liberated, after synthesis but before screening.
Examples of candidate simple libraries which might be
evaluated include derivatives of. the followir~.g:
Cyclic Compounds Containing One Hetero l~tom
Heteronitrogen
pyrroles
pentasubstituted pyrroles
pyrrolidines
pyrrolines
prolines
indoles
beta-carbolines
pyridines
dihydropyrid'ines
1,4-dihydropyridines
pyrido[2,3-d]pyrimidines
tetrahydro-3H-imidazo [4, 5-c] pyridines
Isoquinolines
~tetrahydroisoquinolines
quinolones
beta-lactams
. azabicyclo[4.3.0]nonen-8-one amino acid
Heterooxygen
furans
tetrahydrofurans ,
2,5~disubstituted tet=rahydrofurans
pyrans
hydroxypyranones
.30 tetrahydroxypyranones
'.' gamma-butyrolactones
Heterosulfur
sulfolenes
Cyclic Compounds with Two or More Hetero atoms ' '
' Multiple heteronitrogens '
imidazoles
pyrazoles
piperazines
-diketopiperazines
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arylpiperazines '
benzylpiperazines
benzodiazepines
1,4-benzodiazepine-2,5-diones
hydantoins
5-alkoxyhydantoins
dihydropyrimidines
1,3-disubstituted-5,6-dihydopyrimidine-2,4-
diones .
cyclic ureas ,
CyCll.C thioureas
quinazolines
chiral 3-substituted-quinazoline-2,4-
diones
triazoles
1,2,3-triazoles
purines ~ ,
Heteronitrogen and Heterooxygen
dikelomorpholines
isoxazoles
isoxazolines
Heteronitrogen and Heterosulfur
thiazolidines . '
N-axylthiazolidines
dihydrothiazoles
2-methylene-2,3-dihydrothiazates
2-aminothiazoles
thiophenes
3-amino thiophenes
4-thiazolidinones
4-melathiazanones
benzisothiazolones , _ ,
For details on synthesis of libraries, see Nefzi, et
al., Chem. Rev., 97:449-72 (1997), and references cited
therein. , ~ .- , '
Pharmaceutical Methods and Prepara'tions-
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The preferred animal subject of the present invention
is a mammal. By the term "mammal" is meant an individual
belonging to the class Mammalia. The invention is
particularly useful in the treatment of human subjects,
5 . although it is intended for veterinary and nutritional uses
as well. Preferred nonhuman subjects are of the orders
Primata (e.g., apes and monkeys), Artiodactyla or
Perissodactyla (e. g., cows, pigs, sheep, horses, goats),
Carnivora (e. g., cats, dogs), Rodenta (e. g., rats, mice,
10 guinea pigs, hamsters), Lagomorpha (e. g., rabbits) or other
pet, farm or laboratory mammals.
The term "protection", as used herein, is intended to
include "prevention,!' "suppression" and "treatment."
"Prevention", strictly speaking, involves administration of
15 ' the pharmaceutical prior to the induction of the disease (or
other adverse clinical condition). "Suppression" involves
administration of the composition prior to the clinical
appearance of the disease. "Treatment" involves
administration of the protective composition after the
20 appearance of the disease.
It will be understood that in human and veterinary
medicine, it is not always possible to distinguish between .
"preventing" and "suppressing" since the ultimate inductive
event or events may be unknown, latent, or the patient is
25 not ascertained until well after the occurrence of the event
or events. Therefore, unless qualified, the term
"prevention" will be understood to refer to both prevention.
in the strict. sense, and to suppression.
The preventative or prophylactic use of a
30 pharmaceutical usually involves identifying subjects who are
at higher risk than the general population of contracting, '
the disease, and administering the pharmaceutical to them in
advance of the.clinical appearance of the disease. .The
effectiveness of such use is measured by comparing the
35 subsequent incidence or severity of.the disease, or of
particular symptoms of the disease, in the'treated subjects
against that in untreated subjects of the.same high risk
group .
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While high risk factors vary from disease to disease,
in general, these include (1) prior occurrence of the
disease in one or more members of the same family, or, in
the case of a contagious disease, in individuals with whom
the subject has come into potentially contagious contact at
a time when the earlier victim was likely to be contagious,
(2) a prior occurrence~of the disease in the subject, (3)
prior occurrence of a related disease, or a condition known
to increase the likelihood of the disease, in the subject;
(4) appearance of a suspicious level of a marker of the
disease,, or a related disease or condition; (5) a subject
who is immunologically compromised; e.g., by radiation
treatment, HIV infection, drug use,, etc., or (6) membership
in a particular group (e. g., a particular age, sex, race,
~15 ethnic group, etc.) which has been epidemiologically
associated with that disease.
In some cases, it may be desirable to provide
prophylaxis for the general population, and not just a high
risk group. This is most likely to be the case when
essentially all.are at risk of contracting the disease, the
effects of the disease are serious, the therapeutic index of
the prophylactic agent is high, and the cost of the agent is
low.
. A prophylaxis or treatment may be curative, that is,
directed at the underlying cause of a disease,,or
ameliorative, that is, directed at the symptoms of the
disease, especially those which reduce the quality of~life.
It should also be understood that to be useful, the
protection provided need not be absolute, provided that it
is sufficient to,carry clinical. value. 'An agent which
provides protection to a lesser degree than do competitive
agents may still be of value if the other agents.are
ineffective.for a particular individual, if it can be used
in combination with other agents to enhance the level of '
protections or if it is safer than competitive agents. It is
desirable that there be.a'statistically significant (p=0.05
or less).vimprovement~in the treated subject relative to an
appropriate untreated control, and it is desirable that this
improvement be at least l0%, more preferably at feast 25%,
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still more preferably at least 50%, even more preferably at
least 1000, in some indicia of the incidence or severity of
the disease or of at least one symptom of the disease.
At least one of the drugs of the present invention may
be administered, by any means that achieve their intended
purpose, to protect a subject against a disease or other
adverse condition. The form of administration may be
systemic or topical. For example, administration of such a
composition may be by various parenteral routes such as
subcutaneous, intravenous, intradermal, intramuscular,
intraperitoneal, intranasal, transdermal, or buccal routes.
Alternatively, or concurrently, administration may be by the
oral route. Parenteral administration can be by bolus
injection or by gradual perfusion over time.
A typical regimen comprises administration of an
effective amount of the drug, administered over a period
ranging from a single dose, to dosing over a period of
hours, days, weeks, months, or years.
It is understood that the suitable dosage of a drug of
the present invention will be dependent upon the age, sex,
health, and weight of the recipient, kind of concurrent
treatment, if any, frequency of treatment, and the nature of
the effect desired. However, the most preferred dosage can
be tailored to the individual subject, as is understood and
determinable by one of skill in the art, without undue
experimentation.. This will typically involve adjustment of
a standard dose, e.g., reduction of the dose if the patient
has a low body weight.
Prior to use in humans, a drug, will first be evaluated
for safety and efficacy in. laboratory animals. In human'
clinical, studies, one would begii~.,with a dose expected to be
safe in humans, based~,on'the preclinical data'for the drugs'
in question, and on customary doses for analogous drugs (if
any). If this dose~is effective,:the dosage may be
decreased, to determine the minimum effective dose, if ~ '
'desired. '~If this dose is ineffective; it will be cautiously
increased, with the patients monitored for~'_signs offside
effects. See, .e.g. ,'~' Serkoui et al, ~ eds. , The Merck, Manual,
l5th edition, ~ Merck and , Co. , Rahway, , N. J. , 1987; . Goodman ~.et~
'~ , . ~ . '
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al., eds., Goodman and Gilman.'s The Pharmacological Basis of
Therapeutics,. 8th edition, Pergamon Press, Inc., Elmsford,
N.Y., (1990); Avery's Drug Treatment: Principles and
Practice of Clinical Pharmacology and Therapeutics, 3rd
edition, ADIS Press, LTD., Williams and Wilkins, Baltimore,
MD. (1987), Ebadi, Pharmacology, Little, Brown and Co.,
Boston, (1985), which references and references cited
therein, are entirely incorporated herein by reference.
The total dose required for each treatment may be
administered by multiple doses or in a single dose. The
protein may be administered alone or in conjunction with
other therapeutics,directed to the disease or directed to
other symptoms thereof. '
Typical pharmaceutical doses, for adult humans, are in
the range of 1 ng to 10g per day,:more often 1 mg to 1g per
day.
The appropriate dosage form will depend on the disease,
the pharmaceutical, and the mode of administration;
possibilities include tablets, capsules, lozenges, dental
pastes, suppositories, inhalants, solutions, ointments and
parenteral depots. See, e.g., Berker, supra, Goodman,
supra, Avery, supra and Ebadi, supra, which are entirely
incorporated herein by reference, including all references
cited therein.
In the case of peptide drugs, the drug maybe
administered in the form.of an expression vector comprising '
a nucleic acid encoding the peptide; such a vector, after
incorporation into'the genetic complement of a cell of the
patient, directs synthesis of the peptide. Suitable vectors
include genetically engineered poxviruses (vaccinia),
adenoviruses, adeno-associated viruses, herpesviruses and
lentiviruses which are or have been rendered nonpathogenic.
In addition to at least one drug as described herein,,a
pharmaceutical composition may contain suitable
35pharmaceutically acceptable carriers, such as excipients.',
carriers and/or auxiliaries which facilitate processing of
the active compounds into preparations which can be'used
pharmaceutically. See, e.g., Berker, supra, Goodman, ,supra;
Avery, supra and Ebadi,supra, which are entirely
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incorporated herein by reference, included all references
cited therein.
Assay Compositions and Methods
Tar ea t Organism
The invention contemplates that it may be appropriate
to ascertain or to mediate the biological activity of a
substance of this invention in a target organism.
The target organism may be a plant, animal, or
microorganism.
In the case of a plant, it may be an economic plant, in
which case the drug may be intended to increase the disease,
weather or pest resistance,'alter the growth ,
characteristics, or otherwise improve the useful'
characteristics or mute undesirable characteristics of the
plant. Or it may be a weed, in which case the drug may be
intended to kill or otherwise inhibit the growth of the
plant, or to alter its characteristics to convert it from a
weed to an'economic plant. The plant may be a tree, shrub,
crop, grass, etc.: The plant may be an algae (which are in
some cases also microorganisms), or a vascular plant,
especially gymnosperms (particularly conifers) and
angiosperms. Angiosperms may be monocots or dicots. The
plants of greatest interest are rice, wheat, corn, alfalfa,
soybeans, potatoes, peanuts, tomatoes, melons, apples,
pears, plums, pineapples, fir, spruce, pine, cedar, and oak.
If the target organism is.a microorganism, it may be.
algae, bacteria, fungi, or a virus (alth.ough the biological
activity of a.virus must be determined in a virus-infected
cell). The microorganism may be human or other animal or
plant pathogen, or it may be nonpathogenic. It may be a
soil or water organism; or one which normally liveslinside
other living things:
If the target organism is an animal, it may be a' .
vertebrate or a nonvertebrate animal. Nonvertebrate animals
are chiefly of interest when they act as pathogens or
parasites, and.the drugs are intended to act as biocidic or
biostatic agents. Nonvertebrate animals of :interest include '
worms, mollusks, and arthropods. '
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The target organism may also be a vertebrate animal,
i.e., a mammal, bird, reptile, fish or amphibian. Among
mammals, the target animal preferably belongs to the order
Primata (humans, apes and monkeys), Artiodactyla (e. g.,
5 cows, pigs, sheep, goats, horses), Rodenta (e. g., mice,
rats) Lagomorpha (e. g., rabbits, hares), or Carnivora (e. g.,
cats, dogs). Among birds, the target animals are preferably
of the orders Anseriformes (e.g., ducks, geese, swans) or
Galliformes (e.g., quails, grouse, pheasants, turkeys and
10 chickens). Among fish,~the target animal is preferably of
the order Clupeiformes (e. g., sardines, shad, anchovies,
whitefish, salmon).
Target Tissues
15 The term "target tissue" refers to any whole animal,
physiological system, whole organ, part of organ,
miscellaneous tissue, cell, or cell component (e.g., the
cell membrane) of a target animal in which biological
activity may be measured.
20 Routinely in mammals onewwould choose to Compare and
Contrast the biological impact on virtually any and all
tissues which express the subject receptor protein. The
main tissues to use are: brain, heart, lung, kidney, liver,
pancreas, skin, intestines, adipose,. stomach, skeletal
25 muscle, adrenal glands, breast, prostate, vasculature,
retina,- Cornea, thyroid gland, parathyroid glands, thymus,
bone marrow, bone, etc.
Another Classification would be by'cell type: B Cells,
T cells, macrophages, neutrophils, eosinophils,. mast Cells,
30. platelets,,megakaryocytes, erythrocytes, bone marrow.stomal
Cells, fibroblasts, neurons, Iastrocytes, neuroglia,,
microglia, epithelial cells (from any organ, e~.g. skin,
breast, prostate, lung; intestines etC), Cardiac muscle
cells, smooth muscle cells,'striated muscle Cells, v
35 osteoblasts, oste~oCytes, Chondroblasts; Chondrocytes,
keratinocytes, melanocytes, etc.' . ~. , '
Of course, in the Case of a unicellular organism, there
is no distinction between the."target organism" and the
°target tissue.
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Screening Assays
Assays intended to determine the binding or the
biological activity of a substance are called preliminary
screening assays.
Screening assays will typically be either in vitro
(cell-free)'assays (for binding to an immobilized receptor)
or cell-based assays (for alterations in the phenotype of
the cell). They will not involve screening of whole
multicellular organisms, or isolated organs. The comments
on diagnostic biological assays apply mutatis mutandis to
screening cell-based assays.
In Vitro vs. In Vivo Assays
The term in vivo is descriptive of an event, such as
binding or enzymatic action, which occurs within a living
organism. The organism in question may, however, be
genetically modified. The term in vitro refers to an event
which occurs outside a living organism. Parts of an
organism (e.g., a membrane,~or an isolated biochemical) are
used, together with artificial substrates and/or conditions.
For the purpose of the present invention, the term in vitro
excludes events occurring inside or on an intact cell,
whether of a unicellular or multicellular organism.
~25 In vivo assays include both cell-based assays, and '
organismic assays,.~The cell-based assays include both assays
on unicellular organisms, and assays on isolated cells or
cell cultures derived from.multicellular organisms. The / '
cell cultures may be mixed, provided that they are not ,
organized into tissues or organs. The term organismic assay,
' refers to assays on whole multicellular organisms, and
assays on isolated organs or tissues of such organisms.
In vitro Diagnostic Methods and Reagents
,
~.
' , The in vitro assays of the present iriyention maybe ~ ' .'
applied to any suitable analyte-containing sample, and may
be qualitative, or quantitative in nature.
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Samp1 a
The sample will normally be a biological fluid, such as
blood, urine, lymph, semen, milk, or cerebrospinal fluid, or
a fraction or'derivative thereof, or a biological tissue, in
the form of, e.g., a tissue section or homogenate. However,
the sample conceivably could be (or derived from) a food or
beverage, a pharmaceutical or diagnostic composition, soil,
or surface or ground water. If a biological fluid or
tissue, it may be taken from a human or other mammal,
vertebrate or animal, or from a plant. The preferred sample
is blood; or a fraction or derivative thereof.
Binding and Reaction Assays .
The assay may be a binding assay, in which one step
involves the binding of a diagnostic reagent to the analyte,
or a reaction assay, which involves the reaction of a
reagent with the analyte., The reagents used in a binding
assay may be classified as to the nature of their
interaction with analyte: (1) analyte analogues, or (2)
analyte binding.molecules (ABM). They may be labeled or-.
insolubilized.
. In a reaction assay, the assay may look for a direct
reaction between the analyte and a reagent which is reactive
' with the analyte, or if the analyte is an enzyme or enzyme
inhibitor,.for a reaction catalyzed or inhibited by the
analyte. The reagent~may be a reactant, a catalyst, or an~
inhibitor fox the reaction.
An assay may involve a cascade of steps in which the
product of one step acts as the target for the next step.
These steps may be binding steps, reaction steps, or a
combination thereof.
,'
Signal Producing System (SPS)
In order to detect'the presence, or measure the amount,
of an analyte, the assay must provide:for a signal producing
systeriz (SPS) in which there is a detectable difference in
the signal producecl, depending on whether the analyte is'
present or absent (or,.in a quantitative assay, on the
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amount of the analyte). The detectable signal may be one
which is visually detectable, or one detectable only with
instruments. Possible signals include production of colored
or luminescent products, alteration of the characteristics
{including amplitude or polarization) of absorption or
emission of radiation by an assay component or product, and
precipitation or agglutination of a.component or product..
The term "signal" is intended to include the discontinuance
of an existing signal, or a change in the rate of change of
an observable parameter, rather than a change in its
absolute value. The signal may be monitored manually or
automatically.
In a reaction assay, the signal is often a product of
the reaction. In a binding assay, it is normally provided
by a label borne by a:labeled reagent.
Label s
The component of the signal producing system which is
most intimately associated with.the diagnostic reagent is
called the'"label". A label may be, e.g., a radioisotope, a
fluorophore, an enzyme, a co-enzyme, an enzyme substrate, an
electron-dense compound, an agglutinable particle.
The radioactive isotope can be detected by such means
as the use of.a gamma counter or a scintillation counter or
by autoradiography. Isotopes which are particularly useful
for the purpose of the present °invention include 3H, '-25I,
isil~ 3ss~ i4C~ aap and asp. ~zsl is preferred for antibody
labeling. v ;,
The label may also be a fluorophore. When the
~ fluorescently labeled reagent is exposed to light of the
proper GSave length, its presence can then be detected due to ,
fluorescence. ,Among the most commonly used~.fluorescent
labeling compounds are fluorescein isothiocyanate,
rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-
phthaldehyde and.fluores'camine.
Alternatively, fluorescence-emitting metals such as
,, izsEu, or others of the lanthanide series, may be
incorporatedinto~a diagnostic reagent using such metal
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chelating groups as diethylenetriaminepentaacetic acid
(DTPA) of ethylenediamine-tetraacetic acid (EDTA).
The label may also be a chemiluminescent compound. The
presence of the chemiluminescently labeled reagent is then ,
.determined by detecting the presence of luminescence that
arises during the course of a chemical reaction. Examples
of particularly useful chemiluminescent labeling compounds .
are luminol, isolumino, theromatic acridinium ester,
.imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used for
labeling. Bioluminescence is a type of chemiluminescence:
found in biological systems in which a catalytic protein
increases the,efficiency of.the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by
detecting the presence of luminescence.' Important
bioluminescent compounds for purposes of labeling are
luciferin, luciferase and aequorin.
Enzyme labels, such as horseradish peroxidase and
alkaline phosphatase, are preferred., When an enzyme label
is used, the signal producing system must also include a
substrate for the enzyme. If the enzymatic reaction product .
is not itself detectable, the SPS will include one'or more
additional reactants so that a detectable,product appears.
An enzyme analyte may~,act as its own label if an enzyme
inhibitor is used as a diagnostic reagent.
Binding Assay Formats
Binding assays may be divided into two basic types, v
heterogeneous and homogeneous. In heterogeneous assays, the
interaction between the affinity molecule and the analyte~'
does'not affect'the label, hence, to determine the amount or
presence of analyte, 'bound label, must be separated from free
label. In homogeneous assays, the interaction does affect
the activity of the label,. and therefore analyte levels can
be deduced without the need for a'separation step.
Tn one'embodiment, the ABM is insolubilized by coupling
it to a rriacromolecular support, 'and analyte in the'sample,is
allowed to 'cotripete with a known quantity of. ~a ''labeled or
specifically,labelable,analyte analogue. 'The "analyte,,
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analogue" is a molecule capable of competing with analyte .
for blinding to the ABM, and the term is intended to include
analyte itself. It may be labeled already, or it may be
label ed subsequently by specifically binding the label to a
5 , moiety differentiating the analyte analogue from analyte.
The solid and liquid phases are separated, and the labeled
analyte analogue in one phase is quantified. The higher the
level of analyte analogue in the solid phase, i.e.,
sticking to the ABM, the lower the level of analyte in the
10 sample.
In a "sandwich assay", both an insolubilized ABM, and~a
label ed ABM are employed. The analyte is captured by the
insolubilized ABM and is tagged by the labeled ABM, forming.
a ternary complex. The reagents may be added to the sample
15 ~in either order, or simultaneously. The ABMs may be the
same or different. The amount of labeled ABM in.the ternary
compl ex is directly proportional to the amount of analyte in
the s ample. ,
The two embodiments described above are both
20 heterogeneous assays. However, homogeneous assays are
conceivable. The key is that the label be affected by
w~.ether or not the complex is formed.
Conjugation Methods
A label may be conjugated, directly or indirectly , ~ ',
25 (e. g., through a labeled anti-ABM antibody), covalently
' (e.g., with SPDP) or noncovalently,lto the ABM, to produce a
diagnostic reagent. Similarly, the ABM may be conjugated to
~a sol id phase support to form a solid phase ("capture")
diagnostic reagent.
30 Suitable supports include glass, polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases;v
natural and modified celluloses, polyacrylamides, agaroses,
and magnetite The nature of the carrier can be~either ,
soluble to some extent or insoluble for the purposes of the
35 present invention. ' ' ,
The support material may have virtually any possible ,
structural configuration so long as the coupled molecule, is
;.
capable of binding to its target. Thus the support : ,
configuration may be spherical, as in a;bead, or
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cylindrical, as in the inside surface of a test tube,, or the
external surface of a rod. Alternatively, the surface may
be flat such as a sheet, test strip, etc.
Biological Assays
A biological assay measures or detects a biological
response of a biological. entity to a substance.
The biological entity may be a whole organism, an
isolated organ or tissue, freshly isolated cells, an
immortalized cell .line, or a subcellular component (such as
a membrane; this term should not be,construed as including
an isolated,receptor). The entity may be, or may be derived
from, an organism which occurs in nature, or which is
modified irm some way: Modifications may be genetic
(including radiation and chemical mutants, and genetic
engineering) or somatic (e. g., surgical, chemical; etc.).
In the case of a multicellular entity, the modifications may
affect.some or all cells. The entity need not be the target
organism, or a derivative thereof, if there is a reasonable
correlatiori between bioassay activity in the assay entity
and biologi cal activity in the target organism.
The eritity is placed in~a particular environment, which
may be, more or less natural. For example, a culture medium
may, but ne ad not, contain;serum or serum substitutes, and
it may, but-need'not, include a support matrix of some kind,
it may be s t ill, or agitated. 2t may contain particular
biological or chemical agents, or. have particular physical
parameters (e. g.; temperature);, that are intended to nourish
or challenge the biological entity.
There must also be a detectable biological marker for , '
the respons a. At the cellular level, the most common
markers'are cell survival and proliferation,.cell behavior
(clustering, motility), cell morphology (shape, color), and
biochemical activity (overall DNA synthesis, overall protein
:35 , synthesis, anal specific.metabolic activities, such as
utilization of particular nutrierits,~ e.g.,~consumption~of
oxygen, production of, CO2, 'production of organic acids, , '
uptake or c3.ischa'rge of ions) .
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The direr t signal produced by the biological marker may
be transformed by a signal producing system into a different
signal which i s more observable, for example, a fluorescent
or colorimetri c signal.
The entity, environment, marker and signal producing
system are chosen to achieve a clinically acceptable level
of sensitivity, specificity and accuracy. .
In some c aces, the goal will be to identify substances
which mediate the biological activity of a natural
biological entity, and the assay is carried out directly
with that entity. In other cases, the biological entity is
used simply as a model of some more complex {or otherwise
inconvenient t o work with) biological entity. In that
event, the model biological entity is used because activity
in the model system is considered more predictive of
activity in the ultimate natural biological entity than is
simple binding activity in an in vitro system. The model
entity is used instead of the.ultimate entity because the
former is more expensive or slower to work with, or because
ethical considerations forbid working with the ultimate
entity yet. ~ ,
The model entity may be naturally occurring, if the
model entity usefully models the ultimate entity under some
conditions. Or it may be non-naturally occurring, with
modifications that increase its resemblance to the ultimate
entity. ,
Transgeni c animals, such as transgenic mice, rats, and
rabbits, have been'found useful as model systems.
In cell-based model assays, where the biological
activity is mediated by binding toga receptor (target
protein), the receptor may be functionally connected to: a.
signal. (biological marker) producing systerci,' which', may be
endogenous or exogenous,to the cell.
There are a number of techniques of doing this.
~ , .
"hero-Hybrid" Systems ,
In these systems, the binding; of apeptide to the,
,,
target protein results in ascreenable or.selectable~.
phenotypic change, without resort to fusing the target ~~
~, ,.
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protein (or a ligand binding moiety thereof) to an
endogenous protein. It may be that the target protein is
endogenous to the host cell, or is substantially identical
to an endogenous receptor so that it can take advantage of
the letter's native~signal transduction pathway. Or
sufficient elements of the signal transduction pathway
normally associated with the target protein may be
,. engineered into the cell so that the cell signals binding to
the target protein. '
"One-Hybrid" Systems
In these systems, a chimera receptor, a hybrid of the
target protein and an endogenous receptor, is used. The
chimeric, receptor has the ligand binding characteristics of
the target protein a.nd the signal transduction
characteristics of the endogenous receptor. Thus, the
normal signal transd.uction pathway of the endogenous
receptor is subverted.
Preferably, the endogenous receptor is inactivated, or
the conditions of the assay avoid activation of the
endogenous receptor to improve the signal-to-noise ratio.
See Fowlkes USP 5,789,184 for a yeast system. ,
Another type of "one-hybrid" system combines a peptide:
DNA-binding domain fusion with an unfused target receptor
that possesses an activation domain.
"Two-Hybrid" System ,
' : ,~In a preferred embodiment, the cell-based assay is a '
two hybrid system. This teem implies that the ligand is ~ , ,
incorporated into a first hybrid protein, and the receptor ,
into a second hybrid protein. The first hybrid also _
comprises component A of a signal.generating system, and the .
second,hybrid comprises component B of that system.,
Components A. and B, by themselves, are insufficient to
,35 generate a,signal.~~Iowever, if the ligand binds the
receptor, components A and B are brought into sufficiently
close proximity so t hat they can cooperate to generate a
signal, , ' ~ , .
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Components A and B may naturally occur, or be ,
substantially identical t:o moieties which naturally occur,
as components of a singl a naturally occurring biomolecule,
or they may naturally occur, or be substantially identical
to moieties which natural 1y occur, as separate naturally
occurring biomolecules which interact in nature.
Two-Hybrid System: Transcription Factor Type
In a preferred "two - hybrid" embodiment, one member of a
peptide ligand:receptor, binding pair is expressed as a
fusion to a DNA-binding domain (DBD) from a transcription
factor (this fusion prote in is called the "bait"), and the
other is expressed as a fusion to a transactivation domain
(TAD) (this fusion protein is called the "fish"~, the "prey",
or the "catch" ) . The t-ransactivation domain should be
complementary to the DNA- binding domain, i.e., it should
interact with the latter so as to activate transcription of ,
a specially designed reporter gene that carries a binding
site for the DNA-binding domain. Naturally, the two fusion
proteins must likewise be complementary.
This complementarity may be achieved by use of the
complementary and separable~DNA-binding and transcriptional
activator domains of a single transcriptional activator
protein, or one may use complementary domains derived from
different proteins. The domains may be identical to the
native,domains, or mutant s thereof. The assay members may
be fused. directly to the DBD or TAD, or fused through'an
intermediated linker. , ~ ,
' The target DNA operator may be the native operator
sequence, or a mutant opa rator. Mutations. in the operator
may be coordinated with mutations in. the DBD and the TAD.
An example~of a suitable transcription activation system is
one comprising the DNA-binding domain from the bacterial
repressor LexA and the.ac tivation domain from the yeast ,
transcription factor Gal4, with the reporter: gene operably
linked to '.the LexA, operator. ~ '
~, It i;s not necessary to employ the intact target
receptor;. just ~vthe ligand-binding moiety is sufficient.
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The two fusion proteins may be expressed from the same
or different vectors. Likewis e, the activatable reporter .
gene may be expressed from the same vector as either fusion
protein (or both proteins), or from a third vector.
5 Potential DNA-binding domains include Gal4, LexA, and
mutant domains substantially identical to the above:
Potential activation.doma ins include E. coli B42, Gal4
activation domain II, and HSV VP16, and mutant. domains
substantially identical to the above.
10 Potential operators include the native operators for
the desired activation domain, and mutant domains
substantially identical to the native operator
The fusion proteins may comprise nuclear localisation
signals.
15 The assay system will include a signal producing
system, too. The first elemerit of this system is a reporter
gene operably linked to an operator responsive to the DBD
and TAD of~choice. The expres lion of this reporter gene
will result, directly or indirectly, in a selectable or
20 screenable phenotype (the signal). The signal producing
system may include, besides the reporter gene, additional
genetic or biochemical element s which cooperate in the
production of the.signal. Such an element could be, for
example, a selective agent ~in -the cell.growth medium. There
25 may be more than one signal producing system, and,the system
may include more than one reporter gene.
The sensitivity of the system may be adjusted by, e.g.;
use of competitive inhibitors of any step in the activation w
or signal production process; ~..ncreasing or decreasing. the
30, number of operators, using'a stronger or weaker DBD or TAD,
et c . ' , ~, . ' ~ ~ ,
When the signal is the death or survival of the cell in
question; or proliferation or. nonproliferation of the cell
in question, the assay is said to be a selection. When the
35 signal merely results in a det actable phenotype by which the
signaling cell may be ,'differentiated from-the same 'cell in a .',
nonsignaling state ~ (eith.er way being a living cell )' , , the : , ,
assay is a screen.' However, the term "screenin.g assay",may
be used in a:broader sense to iricltide a selection:. When: the'
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96
narrower sense is intended, we will use the term
"nonselective screen".
Various screening and selection systems are discussed
in Ladner, USP 5,198,346.
Screening and selection may be for or against the
peptide: target protein or compound: target protein
interaction.
Preferred assay cells are microbial (bacterial, yeast,
algal, protozooal), invertebrate, vertebrate,(esp.
mammalian, particularly human). The best developed two-
hybrid assays are yeast and.mamrnalian systems.
Normally, two hybrid assays are used to determine
whether a protein X and a protein Y interact, by virtue of
their ability to reconstitute the interaction of the DBD and
, the TAD. However, augmented two-hybrid assays have been
used to detect interactions that depend on a third, non-
protein ligand.
For more guidance on two-hybrid assays, see Brent and
Finley, Jr., Ann. Rev. Genet., 3 1:663-704 (1997); Fremont- w
Ravine, et al., Nature Genetics, 277-281 (16 July 19.97);
Allen, et al., TIBS, 511-16 (Dec. 1995); LeCrenier, et al.,
BioEssays, 20:1-6 (1998); Xu, et al., Proc. Nat.'Aced. sci.
(USA), 94:12473-8 (Nov. 1992); Esotak, et ~al., Mol. Cell.
Biol., 15:5820-9 (1995); Yang, at al., Nucleic Acids Res.,
23:1152-6 (1995); Bendixen, et al., Nucleic Acids Res.,
22:1778-9 (1994); Fuller, et al., BioTechniques, 25:85-92
(July 1998) ; Cohen; et al . , PNAS (USA) 95:14272-7 (1998) ;
,. Kolonin and Finley, Jr., PNAS (USA) 95:14266-71 (1998). See
also Vasavada, et al., PNAS (USA), 88:10686-90(1991)
(contingent replication assay), and Rehrauer, et al., J.
Biol. Chem., 271:23865-73 91996) (LexA repressor cleavage
assay) .
Two-Hybrid Systems: reporter Enzyme type
'In another embodiment,~the components A and B
reconstitute an enzyme which is not a transcription factor..
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As in the last~example, the effect of the
reconstitution of the enzyme is a phenotypic change which
may be a screenable-change, a selectable change, or both.
In vivo Diagnostic Uses
Radio-labeled ABM maybe administered to the human or
animal subject. Administration is typically by injection,
e.g., intravenous or arterial or other means of
administration in a quantity sufficient to permit subsequent
dynamic and/or static imaging using suitable radio-detecting
devices. The dosage i~ the~smalle st amount capable of
providing a diagnostically effective image, and may be
determined by means conventional in the art, using known
radio-imaging agents as a guide.
Typically, the imaging is carried out on the whole body
of the subject, or on that portion of the body or organ
relevant to the condition or disease under study. The
amount of radio-labeled ABM accumulated at .a given point in
time in relevant target organs can then be quantified.
A particularly suitable radio-detecting device is a
scintillation camera, such as a gamma camera. A
scintillation.camera is a stationary device that can be used
to image distribution of radio-labeled ABM. The detection
' device in the~camera senses the radioactive decay, the
distribution of which can be recorded. Data produced by the
imaging system can be digitized.. The digitized information
can be analyzed over time discontinuously or continuously.
The digitized data can be processed to produce images,
called frames, of the pattern of uptake of the radio-labeled
ABM in the target, organ at a discrete point in time. In
most continuous (dynamic) studies, quantitative data is
. obtained'by observing changes in distributi-ons of
radioactive decay in target organs over time. In other
words, a time-activity analysis of .the data will illustrate
~35 uptake through clearance of the radio-labeled binding
protein by the,target organs with time: ' ' . '
Various factors should be tal~en into consideration in
selecting an appropriate radioisotope. The radioisotope
must be',selected with a view. to obtaining good quality
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98
resolution upon imaging, should be safe for diagnostic use
in humans and animals, and should preferably have a short
physical half-life so as to decrease the amount of radiation
received by -the body. The radioisotope use d should
preferably be pharmacologically inert, and., in the
quantities administered, should not have any substantial
physiological effect.
The ABM may be radio-labeled with different isotopes of
iodine, for example '~z32, 12s2, or 1312 (see for example, U.S.
Patent'4,609e725). The extent of radio-lab eling must,
however be monitored, since it will affect the calculations
made based on the imaging results (i.e. a d iiodinated ABM
will result e n twice the radiation count of a similar
monoiodinated ABM over the same time frame) .
In applications to human subjects, it ~rnay be desirable
to use radioisotopes other than lzs2 for labeling in order to
decrease the total dosimetry exposure of the human body and
to optimize t he detectability of the labeled molecule
(though this radioisotope can be used if circumstances
require). Ready availability for clinical use is also a
factor. Accordingly, for human application , preferred
radio-labels are for example, 99'"Tc, 6'Ga, 68Ga, 9°Y, lllln,
113mIn, 1232 ~ iasRe ~ lBeRe or ziiAt .
The radio-labeled ABM may be prepared by various
methods. The se include radio-halogenation by the chl.oramine ,
- T method or the lactoperoxidase method and subsequent
purification by HPLC (high pressure'liquid chromatography),.
for example as described by J. Gutkowska et al in '
"Endocrinology and Metabolism Clinics of America: (1987) 16
(1) :183. Other known, methods of radio-labeling can be used,
such. as IODOBEADST"". ' ,
There era a number of different methods of delivering
the radio-labeled ABM to ,,the end-user. Tt may,be '
administered by any means that enables the active agent to'
reach the agent's site of action in the body of a mammal.
Because proteins are subject to being digest ed. when
administered orally, parenteral administrate on, i:e.,'
intravenous,: subcutaneous, intramuscular, would ordinarily
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be used to optimize absorption of an ABM, such as an
antibody, which is a protein.
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EXAMPhES
100
We are utilizing a~mouse model of diet-induced obesity
that progresses to diabetes. The diet is high in fat and
has been. documented to lead to diabetes in C57BL/6 J mice
(Surwit at al., 1988),. After weaning, C57BL/6J mice were
fed either the high fat diet or a standard lab chow diet for
16 weeks. Body weight was monitored bi-weekly. Fasting
glucose and insulin levels were measured after 2, 4, 8, and
16 weeks on the diets. At each time point, several diabetic
and control mice were sacrificed and a number of t issues
collected. For further analysis, RNA was extracte d from the
gastrocnemius muscles at each time point and used in DNA
microarray analyses.
Animal Models . '
Obesity and subsequent hyperinsulinemia and
hyperglycemia were.induced by feeding a group of 3 week old
mice (50 C57BL/6 males) a high-fat diet (Bio-Serve,
Frenchtown, NJ, #F1850 High Carbohydrate-High Fat; 56% of
calories from fat, 16% from protein and 27% from
carbohydrates): Another group of 3 week old mice (20
C57B1/6 males) were fed the normal control diet (PI~lI
Nutrition International Inc., Brentwood, M0, Prolab RMH3000;
14% of calories from fat, 16% from protein and 60% from
carbohydrates). The mice were placed onto the respective
diets immediately following weaning. Animal weights were
determined weekly. Fasting blood-glucose and plasma insulin
measurements were determined~after 2, 4, 8 and 16 weeks on
the respective diets.
The day after obtaining body weight measurements at the
indicated time points, mice were fasted 8 hours and blood
glucose concentrations were measured via tail blood. samples
using a One,Touch Glucometer (Lifescan). For insul in
measurements, blood was collected into heparinized 'tubes,
plasma obtained by centrifugation~and.insulin concentrations
determined using.an Ultra-Sensitive Rat Insulin ELISA kit
(ALPCO) as instructed by the manufacturer. Values were
' adjusted by a factor of 1.23 as determined by the , .,
manufacturer to correct, for species difference in cross-
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101
reactivity with. the antibody (bottom panel). Results
reflect mean ~ SE of 50 mice on the HF diet and 20 mice on
the Std diet.
Normal weight, normal fasting blood glucose and normal
fasting plasma insulinlevels are defined as the respect ive
mean values of the animals fed the control diet.
Two of the "most typical" animals were selected for
each group (Control, hyperinsulinemic and Diabetic) at a ach
time point ( 2,4, 8, and 16 weeks after commencement,of
diet) for sacrifice. The selected mice were sacrificed and
muscle tissue obtained and immediately processed for RNA_
isolation.
Fasting Blood Glucose Levels. '
Blood glucose levels 'was measured from a drop of b1 ood
taken from the tip of the tail of fasted (8 hr) mice using a
Lifescan,Genuine One Touch. glucometer. All measurements
occurred between 2:00 pm and 5:00 pm.
Plasma insulin measurements.
Blood was collected from the tail of . fasted (8 hr) mice
into a heparinized capillary tube and stored on ice. A11
collections occurred between 2:00 pm and 5:00 pm. Plasma
was separated from red blood cells by centrifugation for 10
minutes at 8000 x g and then stored at~-20'C. Insulin
concentrations were determined using the Rat~Insulin ELISA
kit and rat insulin standards (ALPCO);essentially as
instructed by the manufacturer. Values were adjusted by a
y factor of 1:23'as determined by the manufacturer to correct
for the species difference in cross-reactivity with the
antibody, ~ '. ,
' RNA isolation.,
Total RNA was isolated from muscle (skeletal.muscle,
~35 specifically, gastrocnemius) of two mice at each time po 3nt
during the progression of HF diet-induced type 2 diabete s,
. as well as ,age-matched controls on the'~Std, diet, ;'using the . ' '
"RNA'STAT-60 Total RNA/mRNA Isolation Reagent.according to ;
' the manufacturer's instructibns (Tel-Test; Friendswood, TX).
,, , ,
,, . , ,
~. , , '
,~' ' ' ,
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Sample Quantification and Quality Assessment
Total RNA was quantified and assessed for quality on a
Bioanalyzer RNA 6000 Nano chip (Agilent). Each chip
contained an interconnected set o t gel-filled channels that
allowed for molecular sieving of ~.ucleic acids. Pin-
electrodes in the chip were used -to create electrokinetic
forces capable of driving molecules through these micro-
channels to perform electrophoret~..c separations. Ribosomal
peaks were measured by fluorescence signal and displayed in
an electropherogram. A successful total RNA sample featured
2 distinct ribosomal peaks (18S arld 28S rRNA).
Biotinylated cRNA Hybridization Target.
Total RNA was prepared for use as a hybridization
target as described in the manufacturer's instructions for
CodeLink Expression Bioarrays(TM) (Amersham Biosciences).
The CodeLink Expression Bioarrays utilize nucleic acid
hybridization of a biotin-labeled complementary RNA(cRNA)
target with DNA oligonucleotide probes attached to a gel
matrix.
The biotin-labeled cRNA target is prepared~by a linear
amplification method. Poly (A) + RNA (within the total RNA
population) is primed for reverse transcription by a DNA
oligonucleotide containing a T7 RI3A polymerase promoter 5~
to a (dT) 24 sequence. After secor.~d-strand cDNA synthesis,
the cDNA serves as the template ire an in vitro transcription
(IVT) reaction to produce the target cRNA. The IVT is
performed in the presence of bioti nylated nucleotides to
label the target cRNA.. This procedure results in.a 50-200
fold linear amplification of the input poly (A) + RNA.
' .' '
TIybridization Probes. ~ , '
The oligonucleotide probes~we ~e provided by the
Codelink Uniset Mouse I Bioarray (.~mersham, product'code
300013).~Amine-terminated oligonuc ~ eotide probes are
attached to a three-dimensional ~ po~.ya,crylamide gelvmatrix:
There,are 10,000 oligonucleotide p~obes,'each specific to.a ~.
well-characterized mouse~gene.rEac~. rnouse.gene is '..
y ''
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103
representative of a unique gene cluster from the fourth
quarter 2001 Genbank Unigene build. There are also 500
control probes.
The sequences of the probes are prop rietary to
Amersham. However, for each probe, Amers ham identifies the
corresponding mouse gene by NCBI accessio n number, OGS,
LocusLink, Unigene Cluster ID, and description (name).
This information should be available from Amersham. In the
case of the differentially expressed probes, this
information is duplicated in master table 1. For the
complete list, see
. http://www4.amershambiosciences.com/aptri.~/upp01077.nsf/Cont
ent/codelink literature
Under "Gene Lists", select "Uniset Mouse I", and a gene
list, in Excel format, can be downloaded.
Hybridization
Using the cRNA target, the hybridiza Lion reaction
mixture is prepared and loaded into array chambers for
bioarray processing as set forth in the manufacturer's
instructions for CodeLink Gene Expression BioarraysTM
(Amerhsam Biosciences). Each sample is hybridized to an
individual microarray. Hybridization is at 37°C. The
hybridization buffer.is prepared as set firth in the
Motorola instructions.,.Hybridization to the microarray is ,
detected with an avidinated fluorescent r agent,
Streptavidin-.Alexa Fluor ~ 647 (Amersham) .
Mouse Gene Expression Analysis
Processed arrays were scanned using a GenePix 4000B
~Microarray Scanner (Axon Instruments, Inc .); array images
were acquired using the,Amersham CodeLinkT"" Analysis Software
(Release 2.2). The Amersham CodeLinkTT'' Ana lysis Software '
gives an integrated optical density (IOD) value for every
;spot;, a~unique background value for that spot is subtracted, ~ '
resulting in "raw". data points. Individual chips , are then
normalized by the Amersham CodelinkT"' software according to
the median~..raw intensity for all 10,000 genes. A negative
. . .
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104
control threshold f0.2) is also calculated according to the
control probes. The expression data wa ~ analyzed to identify
genes whose expression levels changed significantly with
respect to: ,
Normal mice compared to hyperinsu 1 inemic mice at 2, 4,
8 and 16 weeks on normal vs. high -fat diet.
Normal mice compared to hyperinsu 1 inemic/hyperglycemic
mice at 2, 4, 8 and 16 weeks on normal vs. high-fat
diet. .
Hyperinsulinemic compared to
hyperinsulinemic/hyperglycemic mice at 2, 4, 8 and. l6
weeks on high-fat diets.
Database Searches Nucleotide sequences and predicted amino
acid sequences were compared to public domain databases
using the Blast 2.O program (National Center for
Biotechnology Information, National Institutes of Health).
Nucleotide sequences were displayed us~.ng ABI prism Edit
View 1Ø1 (PE Applied Biosystems, Foster City, CA).
Nucleotide database searches were conducted with the
then~current versionof BLASTN 2Ø12, see Altschul, et al.,
"Gapped BLAST and PSI-BLAST: a new generation of protein
'.database search programs",' Nucleic Acids Res., 25:3389-3402
(1997). Searches employed the default parameters, unless,
otherwise stated. ; ''
For blastN,searches, the default was the blastN matrix
(1,-3), with gap penalties of 5 for existence and 2 for
extension. ~'
Protein database searches were conducted with the then-
current version of BLAST X, see Altschul et al. (1997);
' supra. Searches employed the default parameters, unless
otherwise stated. The~scoring matrix was' BLOSUM62, with'gap.
' costs of l1 for existence and l:for extrension.~~The standard
low complexity.filter was used. ~ ~ .~ '
'"ref" ,indicatesthat NCBI' s RefSeq is the , source , ,
database.'. The identifier that follows is 'a RefSeq accession
' .,
., . , , .
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105
number, not a GenBank accession number. "RefSeq sequences
are derived from GenBank and provide non-redundant curated
data representing our current knowledge of known genes. Some
records include additional sequence information that was
never submitted to an archival database but is available in
the literature. A small. number of sequences are provided
through collaboration; the underlying primary sequence data
is available in GenBank, but may not be available in any one
GenBank record. RefSeq sequences are not submitted primary
sequences. RefSeq records are owned by NCBI and therefore
can be updated as needed to maintain current annotation or
to incorporate additional sequence information." Sae also
http://www.ncbi.nlm.nih.aov/LocusLink/refseq html
It will be appreciated by those~in the art thatr the
exact results~of a database search will change from day to
day, as new sequences are added. Also, if you query with a
longer version of the original sequence, the results will
change. The results given here were obtained at one time
and no guarantee is made that the exact same hits would be
obtained in a~search on the filing date. However, i f an
alignment between a particular query sequence and a
particular database sequence is discussed, that alignment
should not change (if the parameters and sequences remain
unchanged) .
Northern Analysis.
Northern analysis may be used to confirm the re cults.
,Favorable and unfavorable genes, identified as described
,,above, or fragments thereof, will be used as probes in
Northern'hybridization.analyses to confirm their
differential expression.. Total RNA isolated from subject
mice will be resolved by agarose gel electrophoresis through
a 1% agarose, 1 o formaldehyde denaturing gel, transferred
. to positively charged nylon membrane, and hybridized to a
probe labeled with [32P] dCTP that was'generated from the
aforementioned gene or fragment using the Randotri Primed DNA
".
Labeling Kit'(Roche, Palo Alto, CA), or to a.probe L abeleel
with digoxigenin (Roche Molecular~;Biochemicals,
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Indianapolis, IN), according to~the manufacturer's
instructions.
Real-Time RNA Analysis.
~ Real-time RNA analysis may also be used for
confirmation. For "real-time" RNA analysis, RNA will be
converted to cDNA and then probed with gene-specific primers
made for each clone. "Real-time" incorporation of
fluorescent dye will be measured to determine the~amount of
specific transcript present in each sample. Sample
differences (control.vs. hyperinsulinemic, hyperinsulinemic
vs. diabetic, or control vs. diabetic) wiL 1 be evaluated.
Confirmation using several independent aniir3.als is desirable.
In situ Hybridization
Another form of confirmation may be prov3.ded by
nonisotopic in, situ hybridizations (NISH) vn selected human
(obtained by Tissue Informatics) and mouse tissues using
cRNA probes generated from mouse genes found to be up- or
down-regulated during the disease progression. In situ
hybridizations may also be performed on mou se tissues using'
cRNA probes generated,from differentially expressed DNAs.
These cRNA's will hybridize to their corresponding messenger
RNA's present in cells andvwillvprovide infarmation
° regarding the particular cell types within a. tissue that.is ,
expressing the particular gene as well as tie relative level
of gene expression. The cRNA probes may be 'generated by'in ' ,
vitro transcription of template cDNA by Sp6_ or T7 RNA
polymerase in the presence of digoxigenin-1s-UTP (Roche
Molecular Biochemicals,' Mannheim, Germany; ~ardue, M.L. ';
1985. In: In situ hybridization, Nucleic acid
hybridization, a practical approach: GIRL Press,'Oxford, 179-
202) . . ~,
Transgenic Animals.
Transgenicexpression may be used to confirm thev.results. W
In one embodiment,~a mouse is~engineered to ~overexpress the '
favorable or unfavorable moose gene in ques~ioi~.. , In another '
embodiment, a mouse is engineered to express the . ~ ~ ' .
., , ' . ~ , .
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corresponding favorable or unfavorable human gene. T~. a
third embodiment, a nonhuman animal other than a mouse, such
as a rat, rabbit, goat, sheep or pig, is engineered to
express the favorable or unfavorable mouse or human gene.
I3yperquantitative Tissue Analysis
In addition to gene expression analysis the tissue
sections can also be analyzed using TissueInformatics~
Int.'s TissueAnalytics~ software. A single representative
section may be cut from each tissue block, placed on a
slide, and stained with H&E. Digital images of each, slide .
may be acquired using an research microscope and dig~.tal
camera (Olympu,s E600 microscope and Sony DKC-ST5). TL~.ese
images may be acquired at 20x magnification with a
resolution of 0.64 mm/pixel. A hyperquantitative analysis
may be performed on the resulting images: First a dig.~tal
image analysis can identify and annotate structural objects
in a tissue using machine vision. These objects, which are
constituents of the tissue, can be annotated because trhey
are visually identifiable and have a biological meaning.
Subsequently a quantification of these structures regarding'
their geometric properties like area or stain intensitries
and their relationship to the field of view or per unit area
in terms of a % coverage may be performed. Features o~
parameters for hyper-quantification are specific for each
tissue, and, may also include relations between features, ,
measures of, overall heterogeneity, including orientation,
relative locations, and textures.
'30 Correlation Analysis . ,,
Mathematical statistics provides a rich set of addi-~ional
tools to analyze time resolved data sets of hyper-
quantitative and gene expression profiles for similarL ties,
including rank corre,lation,~the calculation of , regression '
and correlation coefficients, and clustering. Continuous
functions may also be fitted through'the data points~o f
individual'gene and tissue feature data. Relation'bet~een
gene expression and hyper-quantitative tissue data.may- be
' ~; , ,. .
. . . ,
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linear or non-linear, in synchronous or asynchronous
arrangements. .
Example 1
Obesity is increasing at an alarming'rate in the United
States. In parallel, the incidence of type II diabetes. is
also rising. We are interested in defining alterations in
gene expression that correlate with the development of these
conditions in the hopes of reversing these dangerous trends.
Insulin plays a major role in regulating blood glucose
levels. It stimulates the uptake of glucose in adipose
tissue and striated muscle for storage as intracellular
triglycerides and glycogen. Insulin also,inhibits the
release'of glucose from the liver. Normally, this would
prevent the rise in blood sugar concentration that occurs
after eating. However, in the early stages of type 2
diabetes, resistance to insulin is seen:
Muscle plays a major role in glucose metabolism. Thus, it
also is a major contributor to the development of type 2
diabetes. In normal situations, muscle cells respond to
increasing levels of insulin by increasing glucose uptake
from the bloodstream. However, during the very early stages
of~type 2 diabetes, muscle tissue becomes resistant to
insulin, requiring the pancreatic beta cells to increase
insulin secretion. Eventually, the beta cells become unable
to compensate for this increasing insulin resistance from
muscle and other cells, and insulin production drops. Thus,
clinical type 2 diabetes results from the combination of
insulin resistance and impaired beta cell function.
Defects in musclelglycogen synthesis are known to play a
,role in the development of insulin resistance'(Petersen and
Shulman, 2002). At least three steps - .those mediated by
glycogen synthase, hexokinase, and GLUT4 - have, been
reported to be defective. in patients with, type 2 diabetes.
Fatty acids~also,can induce insulin resistance,' and it has
'',been suggested that this .,was a,' consequence: of altered
insulin signaling through ~I3-kinase., ,
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We are utilizing a mouse triodel of diet-induced obesity
that progresses to diabetes. The diet is high in fat, an
increasing component in the U.S. diet, and has been
documented to lead to diabetes in C57BL/6J mice (Surwit et
al., 1988). After weaning, C57BL/6J mice were fed either
the high fat diet or a standard lab chow diet for 16 weeks.
Body weight was monitored bi-weekly. Fasting glucose and .
insulin levels were measured after 2, 4, 8, and 16 weeks on
the diets.
Consumption of the HF diet resulted in significant, .
progressive increases in body weight and fasting insulin
levels in comparison to consumption of the Std diet.
Fasting glucose levels of mice on the HF diet were
dramatically increased at the first time point assayed (2
weeks) and remained high through the duration ~of the
experiment (16 weeks).
At each time point, several diabetic and control mice were
sacrificed and a number of tissues collected. RNA was
extracted from the gastrocnemius muscle at each time point.
In order to identify additional muscle genes involved in
the development of type 2 diabetes, we used microarray
analysis to compare RNA expression levels of 10,000 genes in
muscle of high fat diet fed and control diet fed triice at
various time points,in~the progression of type'2 diabetes.
Microarray analysis provides a more global picture, of gene
regulation, allowing the identification of families or
groups.of genes showing similar expression patterns that
potentially imply~similar or coordinated roles in disease
progression..
. Consumption of .the HF diet resulted in significant,
progressive increases in body weight' and fasting insulin.
levels in comparison o consumption of the~~Std diet.
Fasting glucose levels of mice on the HF diet were
dramatically increased at the first time point assayed (2
weeks) and remained high through the duration of.the
experiment (16 weeks).. ~ ' '.
Of 10,000 genes analyzed, 121 were up-regulated but only 7 .~ ,
down-regulated greater than two-fold in~the diabetic'
' ,'
,, ;
' , : . . '
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110
relative to non-diabetic mice. These genes are listed in
. Master Table 1.'
This distribution of up- and down-regulated genes was much
different from that seen for other organs (liver, pancreas,
and white adipose tissue) where there was a much closer
balance between the number of up-. and down-regulated genes.
Actin, alpha, cardiac (Actcl; NM 009608) was one of the most
down-regulated genes when comparing HF to Std mice. It was
consistently expressed at lower levels in the HF.diabetic
mice in comparison to the Std mice and also steadily
decreased over the 16 week study.
Example 2
Interestingly, further analysis of the time points and
exploration of gene pathways and functionally related genes
revealed a subset of actin-related and actin-binding genes
exhibiting a consistent decrease in expression (although
less than two-fold) im the diabetic mice; 9 of 37
functionally related genes were decreased in'diabetic muscle
at all four time points and an additional 9 were decreased
at three of the four time points. Only two of these genes
had been included in the original list of 7. down-regulated
genes using the two-fold cut-off criterion.
It is possible that this subtle but coordinated down-'
regulation of actin-related or actin-binding genes'reflects
a role.in,the decreased glucose uptake by skeletal muscle
that occurs in diabetes. With nearly half (18 of 37) of the
genes in a related family of genes being consistently down-' ,
regulated in a. study that did not identify a large numberyof
down regulated genes, we feel. that actin and genes in actin-
related pathways may prove to play key roles in muscle as~
obesity and diabetes progress.
The actin-related and actin-binding mouse genes in
question have been,included at the end of Master Table 1,
subtable ' 1A.
. ,
' ' ~ i~ , ' ,
' ,
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Introduction to. Master Tab:Zes
The master tables reflect, applicants' analysis of the gene
chip data.
For each probe corresponding to a differentially expressed
mouse gene, Master Table 1 identifies
Col. 1: The mouse gene (upper) and mouse protein (lower)
database accession #s.
Col. 2: The corresponding mouse Unigene Cluster, as of the
4th Quarter 2001 build.
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Col. 4: A related human protein, identified by its database
accession number. Usually, several such proteins are
identified relative to each mouse gene. These proteins have
been identified by BLAST searches, as explained in cols. 6-
8. y
Col. 5: The name of the related human protein.
Col. 6: The score (in bits) for the alignment performed by
the BLAST program.
Col. 7: The E-value for the alignment performed by the BLAST
program., It is worth noting that Unigene considers a Blastx
E Value of less than 1e-6 to be a "match" to the reference
sequence of a cluster.
Unless otherwise indicated, the bit score and E-value for
the alignment is with respect to the alignment of the mouse
DNA of col. 1 to the human protein of col. 4 by BlastX,
according to the.default parameters.
Master Table,l is divided into three subtables~on the basis
of the behavior in col. 3., If a gene has at least' one
significantly favorable'behavior, and no significantly '
unfavorable ones, it is put into Subtable 1.A. In the
opposite case, it is put. into Subtable 1B. ~If its behavior
is mixed, i.e., at least one significantly favorable~and at
least one significantly unfavorable, it is put into Subtable
a 1C. Note that this'classification is based. on the strongest
observed differential expression behaviors for each of the
three subject comparisons, C-HI, HI-D and C-D.
.;; , ,
, . . . -. . - V 1
The corresponding human'gene clusters'are.also of interest. ~,
'These may be,obtained=in a number of ways. First, one may,
. .
. ,
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search on Unigene
(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene)
for the'identified human protein. Review the "hits" (each
of which is a Unigene record) for those prefixed by "Hs."
Secondly, one may access the Unigene record for the mouse
gene cluster (which is given in Master Table 1), and then
click on "Homologene". This will bring up a new page which
includes the section "Possible Homologous Genes". One of
the entries should be a Homo Sapiens gene (considered by
Unigene to be the most related human gene); click on its ,
Unigene record link.
Additional information of interest may be accessed by
searching with the mouse gene accession # in the Mouse Gene
Informatics database, at http://www.informatics.iax.ora/.
CA 02557181 2006-08-21
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CA 02557181 2006-08-21
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CA 02557181 2006-08-21
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CA 02557181 2006-08-21
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DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 252
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