U.S. patent application number 13/942276 was filed with the patent office on 2014-03-13 for novel targets for treatment of hypercholesterolemia.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. The applicant listed for this patent is Peter J. Tontonoz, Noam Zelcer. Invention is credited to Peter J. Tontonoz, Noam Zelcer.
Application Number | 20140072580 13/942276 |
Document ID | / |
Family ID | 42243346 |
Filed Date | 2014-03-13 |
United States Patent
Application |
20140072580 |
Kind Code |
A1 |
Tontonoz; Peter J. ; et
al. |
March 13, 2014 |
NOVEL TARGETS FOR TREATMENT OF HYPERCHOLESTEROLEMIA
Abstract
In certain embodiments this invention this invention pertains to
the discovery that inhibition of myosin light chain interacting
protein (Mylip) can mitigate one or more symptoms of
hypercholesterolemia. Methods of treating hypercholesterolemia and
methods of screening for agents to treat hypercholesterolemia are
provided.
Inventors: |
Tontonoz; Peter J.; (Pacific
Palisades, CA) ; Zelcer; Noam; (Amstelveen,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Tontonoz; Peter J.
Zelcer; Noam |
Pacific Palisades
Amstelveen |
CA |
US
NL |
|
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
Oakland
CA
|
Family ID: |
42243346 |
Appl. No.: |
13/942276 |
Filed: |
July 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13132296 |
Aug 4, 2011 |
8512964 |
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PCT/US09/67747 |
Dec 11, 2009 |
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13942276 |
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61122273 |
Dec 12, 2008 |
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Current U.S.
Class: |
424/172.1 ;
435/325; 435/369; 435/370; 435/6.12; 435/7.1; 514/44A; 530/389.1;
536/24.5; 800/9 |
Current CPC
Class: |
A01K 2217/052 20130101;
A01K 67/0275 20130101; A01K 2227/105 20130101; A61K 31/713
20130101; G01N 33/5023 20130101; A61P 1/16 20180101; A01K 2267/0375
20130101; A61P 3/06 20180101; A61P 3/10 20180101; A61K 39/3955
20130101; C07K 14/47 20130101; A61K 31/7105 20130101; A01K 67/0276
20130101; C12N 2820/007 20130101; A61P 3/00 20180101 |
Class at
Publication: |
424/172.1 ;
514/44.A; 530/389.1; 536/24.5; 435/325; 800/9; 435/6.12; 435/369;
435/370; 435/7.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/50 20060101 G01N033/50; A61K 31/713 20060101
A61K031/713 |
Goverment Interests
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] This work was supported in part by Grant No: HL066088 from
the National Institutes of Health. The Government has certain
rights in this invention.
Claims
1. A method of inhibiting LDL receptor degradation and/or promoting
LDL uptake in a mammal, said method comprising administering an
agent to said mammal that inhibits expression and/or activity of
myosin light chain interacting protein (Mylip) in an amount
sufficient to inhibit LDL receptor degradation and/or to promote
LDL uptake in said mammal.
2. A method of mitigating one or more symptoms of
hypercholesterolemia in a mammal, said method comprising
administering an agent to said mammal that inhibits expression
and/or activity of myosin light chain interacting protein (Mylip)
in an amount sufficient to mitigate one or more symptoms of
hypercholesterolemia in said mammal.
3. The method of claim 1, wherein Mylip expression and/or activity
is inhibited in the liver of said mammal.
4. The method of claim 1, wherein said mammal is a human.
5. The method of claim 4, wherein said mammal is a human diagnosed
as having or at risk for hypercholesterolemia.
6. The method of claim 1, wherein said agent comprises an siRNA
and/or an shRNA.
7. The of claim 1, wherein said agent comprises a molecule that
binds to the FERM domain of Idol and inhibits interaction or
binding of Idol with LDLR.
8. The method of claim 7, wherein said molecule inhibits
interaction with or binding of Idol to the amino acid residues
conserved between the amino acid residues conserved between LDLR,
VLDLR and apoER2.
9. The method of claim 7, wherein said molecule is an antibody that
binds to the FERM domain of Idol and/or the amino acid residues
conserved between the amino acid residues conserved between LDLR,
VLDLR and apoER2.
10. The of claim 1, wherein said agent is administered in a unit
dosage formulation.
11. The method of claim 1, wherein said agent is combined with an
excipient suitable for administration to a human.
12. The method of claim 11, wherein said excipient is sterile.
13. A composition for inhibiting LDL receptor degradation and/or
promoting LDL uptake in a mammal, said composition comprising an
agent that inhibits Mylip expression and/or activity in a mammal,
wherein said composition is formulated for administration to a
mammal.
14-15. (canceled)
16. The composition of claim 13, wherein said agent comprises an
siRNA and/or an shRNA.
17. The composition of claim 13, wherein said agent comprises
molecule that binds to the FERM domain of Idol and inhibits
interaction or binding of Idol with LDLR
18. The composition of claim 17, wherein said molecule inhibits
interaction with or binding of Idol to amino acid residues
conserved between LDLR, VLDLR and apoER2.
19. The composition of claim 17, wherein said agent comprises an
antibody that binds to the FERM domain of Idol and/or to amino acid
residues conserved LDLR, VLDLR and apoER2.
20. The composition of claim 13, wherein said agent is formulated
with a carrier for administration to a human.
21. The composition of claim 13, wherein said agent is formulated
in a unit dosage formulation.
22. The composition of claim 13, wherein said agent is formulated
as a sterile formulation.
23. A cell transfected with a construct encoding Idol, wherein said
cell expresses Idol at a higher level than the same cell lacking
said construct.
24-25. (canceled)
26. An non-human mammal comprising a cell transfected with a
construct that expresses Idol, whereby said Idol is expressed at a
higher level in a tissue of said animal than the same animal
without said construct.
27. (canceled)
28. A non-human knockout mammal, the mammal comprising a disruption
in an endogenous Mylip/Idol gene, wherein the disruption results in
the mammal exhibiting a decreased level of Idol as compared to a
wild-type mammal.
29-37. (canceled)
38. A method of screening for an agent that inhibits LDL receptor
degradation and/or promotes LDL uptake in a mammal, said method
comprising: contacting a cell with a test agent; detecting the
expression or activity of myosin light chain interacting protein
(Mylip); and scoring a decrease in Mylip expression or activity, as
compared to the expression or activity of myosin light chain
interacting protein (Mylip) in a control as an indication that said
test agent is an agent that inhibits LDL receptor degradation
and/or promotes LDL uptake in a mammal.
39-69. (canceled)
70. A method of screening for an agent that inhibits LDL receptor
degradation and/or promotes LDL uptake in a mammal, said method
comprising: contacting an Idol protein or a fragment thereof
comprising the FERM domain with a test agent; detecting binding of
said test agent to said Idol protein or fragment thereof; and
scoring binding moieties as candidate agents that inhibit LDL
receptor degradation and/or promote LDL uptake in a mammal.
71. A method of screening for an agent that inhibits LDL receptor
degradation and/or promotes LDL uptake in a mammal, said method
comprising: contacting an LDLR or a fragment thereof comprising the
amino acid residues that interact with Idol with a test agent;
detecting binding of said test agent to said LDLR or fragment
thereof; and scoring binding moieties as candidate agents that
inhibit LDL receptor degradation and/or promote LDL uptake in a
mammal.
72. A method of screening for an agent that inhibits LDL receptor
degradation and/or promotes LDL uptake in a mammal, said method
comprising: contacting an LDLR or a fragment thereof comprising the
amino acid residues that interact with Idol and/or an Idol protein
or a fragment thereof comprising the FERM domain with a test agent;
detecting interaction or binding of the LDLR with said Idol protein
or fragment; and scoring moieties that reduce or block LDLR/Idol
interaction or binding as candidate agents that inhibit LDL
receptor degradation and/or promote LDL uptake in a mammal.
73. A kit comprising a container containing a reporter cell wherein
said reporter cell is transfected with a construct expressing an
LDLR, and/or a construct expressing a Mylip.
74-76. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of and priority to U.S. Ser.
No. 61/122,273, filed on Dec. 12, 2008, which is incorporated
herein by reference in its entirety for all purposes.
FIELD OF THE INVENTION
[0003] The present invention relates to the field of lipid
metabolism. In particular, in certain embodiments, this invention
pertains to the discovery that inhibition of myosin light chain
interacting protein (Mylip) can mitigate one or more symptoms of
hypercholesterolemia.
BACKGROUND OF THE INVENTION
[0004] Elevated levels of cholesterol, and particularly of
low-density lipoprotein (LDL) cholesterol, are a major risk factor
for the development of cardiovascular disease, the leading cause of
mortality in the developed world. Current treatments include
lifestyle and dietary changes and the use of oral medication
belonging, mainly, to the statin class of drugs. Statins inhibit
the rate-limiting enzyme in the cholesterol biosynthetic pathway,
HMG-CoA, which results in increased LDL-receptor (LDLR) levels and
clearance of plasma LDL. Despite their widespread use statins
reduce LDL by only 30-50%. Additionally, their use results in
several common side effects including muscle pain, nausea,
diarrhea, or constipation, but in rare cases statins may cause
liver damage or life-threatening rhabdomyolysis. Therefore,
alternatives, or additives to statin treatment would prove
beneficial in treating hypercholesterolemia.
SUMMARY OF THE INVENTION
[0005] It was a surprising discovery that inhibition of that myosin
light chain interacting protein (Mylip) (also known as Inducible
Degrader of the LDLR or "IDOL") expression or activity can inhibit
LDL receptor degradation and/or promote LDL uptake in a mammal,
and/or mitigate one or more symptoms of hypercholesterolemia.
Accordingly, in certain embodiments, methods of achieving these
results are provided. The methods typically involve administering
to a mammal in need thereof an inhibitor of Mylip expression and/or
activity in an amount sufficient partially or fully produces such
results.
[0006] Accordingly, in certain embodiments, methods of inhibiting
LDL receptor degradation and/or promoting LDL uptake in a mammal
are provided. The methods typically involve administering an agent
to the mammal that inhibits expression and/or activity of myosin
light chain interacting protein (Mylip) in an amount sufficient to
inhibit LDL receptor degradation and/or to promote LDL uptake in
the mammal.
[0007] In certain embodiments methods of mitigating one or more
symptoms of hypercholesterolemia in a mammal are provided. The
methods typically involve administering an agent to the mammal that
inhibits expression and/or activity of myosin light chain
interacting protein (Mylip) in an amount sufficient to mitigate one
or more symptoms of hypercholesterolemia in the mammal.
[0008] In certain embodiments the Mylip expression and/or activity
is inhibited in the liver of the mammal. In certain embodiments the
mammal is a human or a non-human. In certain embodiments the mammal
is a human diagnosed as having or at risk for hypercholesterolemia.
In various embodiments the agent comprises an siRNA and/or an
shRNA. In certain embodiments the agent comprises a molecule that
binds to the FERM domain of Idol and inhibits interaction or
binding of Idol with LDLR. In certain embodiments the agent
comprises a molecule that binds to amino acid residues of LDLR that
interact with Idol. In certain embodiments the molecule inhibits
interaction with or binding of Idol to the amino acid residues
conserved between the amino acid residues conserved between LDLR,
VLDLR and apoER2. In certain embodiments the molecule is an
antibody that binds to the FERM domain of Idol and/or the amino
acid residues conserved between the amino acid residues conserved
between LDLR, VLDLR and apoER2. In various embodiments the agent is
administered in a unit dosage formulation. In certain embodiments
the agent is combined with an excipient suitable for administration
to a human. In certain embodiments the excipient is sterile.
[0009] In various embodiments compositions are provided for
inhibiting LDL receptor degradation and/or promoting LDL uptake
and/or for mitigating one or more symptoms of hypercholesterolemia
in a mammal. Illustrative compositions comprise an agent that
inhibits Mylip expression and/or activity in a mammal, where the
composition is formulated for administration to a mammal.
[0010] In certain embodiments the use of an agent that inhibits
Mylip expression and/or activity in the manufacture of a medicament
for inhibiting LDL receptor degradation and/or promoting LDL uptake
and/or for mitigating one or more symptoms of hypercholesterolemia
in a mammal in a mammal is provided. In various embodiments the
composition or use involves an agent that comprises an siRNA and/or
an shRNA. In various embodiments the composition or use involves an
agent that comprises molecule that binds to the FERM domain of Idol
and inhibits interaction or binding of Idol with LDLR. In various
embodiments the composition or use involves an agent comprising a
molecule that inhibits interaction with or binding of Idol to amino
acid residues conserved LDLR, VLDLR and apoER2. In various
embodiments the composition or use involves an agent comprising an
antibody that binds to the FERM domain of Idol and/or to amino acid
residues conserved LDLR, VLDLR and apoER2. In various embodiments
the agent is formulated with a carrier for administration to a
human. In various embodiments the agent is formulated in a unit
dosage formulation. In various embodiments the agent is formulated
as a sterile formulation.
[0011] In certain embodiments non-human mammals are provided
comprising a cell transfected with a construct that expresses Idol,
whereby the Idol is expressed at a higher level in a tissue of the
animal than the same animal without the construct. In certain
embodiments the tissue is the liver of the mammal.
[0012] In certain embodiments viable non-human knockout animals are
provided where the animals comprise a disruption in an endogenous
Mylip/Idol gene, where the disruption results in the mammal
exhibiting a decreased level of Idol as compared to a wild-type
mammal. In certain embodiments the mammal is selected from the
group consisting of a rodent, an equine, a bovine, a porcine, a
lagomorph, a feline, a canine, a murine, a caprine, an ovine, and a
non-human primate. In certain embodiments is a rat or a mouse. In
certain embodiments the disruption is selected from the group
consisting of an insertion, a deletion, a frameshift mutation, a
substitution, and a stop codon. In certain embodiments the
disruption comprises an insertion of an expression cassette into
the endogenous Idol gene. In certain embodiments the expression
cassette comprises a selectable marker. In certain embodiments the
disruption is in a somatic cell. In certain embodiments the
disruption is in a germ cell. In various embodiments the mammal is
homozygous or heterozygous for the disrupted Mylip/Idol gene.
[0013] In various embodiments methods are also provided for
screening for an agent that inhibits LDL receptor degradation
and/or promotes LDL uptake in a mammal, and/or that mitigates one
or more symptoms of hypercholesterolemia. In certain embodiments
the methods involve contacting a cell with a test agent, and
detecting the expression or activity of myosin light chain
interacting protein (Mylip) where a decrease in Mylip expression or
activity, e.g., as compared to the expression or activity of myosin
light chain interacting protein (Mylip) in a control indicates that
said test agent is an agent that inhibits LDL receptor degradation
and/or promotes LDL uptake in a mammal. In certain embodiments the
control comprises a cell contacted with the test agent at a lower
concentration and/or the control comprises a cell that is not
contacted with the test agent. In certain embodiments the detecting
comprises detecting expression of a reporter gene whose expression
is regulated by the myLIP promoter (e.g., by detecting Mylip mRNA
from the cell). In certain embodiments the level of myLip mRNA is
measured by hybridizing the mRNA to a probe that specifically
hybridizes to a myLip nucleic acid. In certain embodiments the
hybridizing is according to a method selected from the group
consisting of a Northern blot, a Southern blot using DNA derived
from a myLip RNA, an array hybridization, an affinity
chromatography, and an in situ hybridization. In certain
embodiments the probe is a member of a plurality of probes that
forms an array of probes. In various embodiments the level of MyLip
mRNA is measured using a nucleic acid amplification reaction (e.g.,
a realtime qPCR). In certain embodiments the amount of MyLip gene
product is detected by detecting the level of MyLip protein from
the cell (e.g., via a method selected from the group consisting of
capillary electrophoresis, a Western blot, mass spectroscopy,
ELISA, immunochromatography, and immunohistochemistry). In certain
embodiments the cell is cultured ex vivo. In certain embodiments
the test agent is administered to an animal comprising a cell
containing a MyLip nucleic acid or a MyLip protein. In various
embodiments detecting the expression or activity of myosin light
chain interacting protein (Mylip) comprises detecting the activity
of Mylip on an LDL receptor. In certain embodiments the cell is a
cell transfected with a construct expressing a detectable LDL
receptor (LDLR) and with a construct expressing an active Mylip. In
certain embodiments the detecting comprises detecting the
detectable LDL receptor where an increase in LDL receptor indicates
inhibition of Mylip. In certain embodiments the detectable LDLR is
an LDLR attached to a detectable label (e.g., an LDLR-luciferase
fusion protein, or an LDLR-GFP fusion protein). In certain
embodiments detecting the expression or activity of myosin light
chain interacting protein (Mylip) comprises detecting Mylip
auto-degredation. In certain embodiments the cell is a cell
transfected with a construct expressing a detectable Mylip. In
certain embodiments an increase of signal from the detectable Mylip
indicates inhibition of Mylip autodegredation activity and
indicates that the test agent is a Mylip inhibitor. In certain
embodiments the detectable Mylip is a Mylip-luciferase fusion
protein, or a Mylip-GFP fusion protein. In certain embodiments the
cell is an HEK293 cell or an HepG2 cell. In certain embodiments
detecting the expression or activity of myosin light chain
interacting protein (Mylip) comprises detecting LDL uptake where an
increase in LDL uptake is an indicator of Mylip inhibition. In
certain embodiments the cell is a cell that expresses LDLR either
endogenously or through transfection. In certain embodiments the
cell is transfected with a construct expression Mylip. In certain
embodiments the cell is contacted with labeled LDL (e.g.,
BODIPY-LDL) and the uptake of the LDL is determined. In certain
embodiments the method is performed in a high throughput format. In
various embodiments the cell is disposed in a microtiter plate. In
certain embodiments the method is performed in a format selected
from the group consisting of a 96 well format, a 100 well format, a
320 well format, a 384 well format, an 864 well format, and a 1536
well format.
[0014] In various embodiments methods are also provided for
screening for an agent that inhibits LDL receptor degradation
and/or that promotes LDL uptake and/or that mitigates one or more
symptoms of hypercholesterolemia in a mammal. The methods typically
involve contacting an Idol protein or a fragment thereof comprising
the FERM domain with a test agent; detecting binding of the test
agent to the Idol protein or fragment thereof; and scoring binding
moieties as candidate agents that inhibit LDL receptor degradation
and/or promote LDL uptake and/or that mitigate one or more symptoms
of hypercholesterolemia in a mammal.
[0015] In various embodiments methods are provided of screening for
an agent that inhibits LDL receptor degradation and/or promotes LDL
uptake in a mammal and/or that mitigates one or more symptoms of
hypercholesterolemia. The methods typically involve contacting an
LDLR or a fragment thereof comprising the amino acid residues that
interact with Idol with a test agent; detecting binding of the test
agent to the LDLR or fragment thereof; and scoring binding moieties
as candidate agents that inhibit LDL receptor degradation and/or
promote LDL uptake and/or that mitigate one or more symptoms of
hypercholesterolemia in a mammal.
[0016] In various embodiments methods are provided of screening for
an agent that inhibits LDL receptor degradation and/or promotes LDL
uptake in a mammal and/or that mitigates one or more symptoms of
hypercholesterolemia. The methods typically involve contacting an
LDLR or a fragment thereof comprising the amino acid residues that
interact with Idol and/or an Idol protein or a fragment thereof
comprising the FERM domain with a test agent; detecting interaction
or binding of the LDLR with the Idol protein or fragment; and
scoring moieties that reduce or block LDLR/Idol interaction or
binding as candidate agents that inhibit LDL receptor degradation
and/or promote LDL uptake and/or that mitigate one or more symptoms
of hypercholesterolemia in a mammal.
DEFINITIONS
[0017] The term microtiter plate refers to an apparatus comprising
a plurality of wells within which can be disposed reagents, and/or
cells, and/or nematodes or other organisms, and the like.
Commercially available microtiter plates are typically commercially
available in 96 well, 100 well, 320 well, a 384 well, 864 well, and
1536 well formats. The microtiter plates can be clear or opaque and
can be fabricated out of low fluorescence materials.
[0018] A "genetic knock out" refers to an organism in which the
normal activity/expression of one or more genes is
disrupted/inhibited.
[0019] A "test agent" refers to refers to an agent that is to be
screened in one or more of the assays described herein. The agent
can be virtually any chemical compound. It can exist as a single
isolated compound or can be a member of a chemical (e.g.
combinatorial) library. In a particularly preferred embodiment, the
test agent will be a small organic molecule.
[0020] The term small organic molecules refers to molecules of a
size comparable to those organic molecules generally used in
pharmaceuticals. The term excludes biological macromolecules (e.g.,
proteins, nucleic acids, etc.). In certain embodiments preferred
small organic molecules range in size up to about 5000 Da, more
preferably up to 2000 Da, and most preferably up to about 1000
Da.
[0021] As used herein, an "antibody" refers to a protein consisting
of one or more polypeptides substantially encoded by immunoglobulin
genes or fragments of immunoglobulin genes. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon and mu constant region genes, as well as myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0022] A typical immunoglobulin (antibody) structural unit is known
to comprise a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these regions of the light and heavy chains
respectively.
[0023] Antibodies exist as intact immunoglobulins or as a number of
well characterized fragments produced by digestion with various
peptidases or expressed de novo. Thus, for example, pepsin digests
an antibody below the disulfide linkages in the hinge region to
produce F(ab)'.sub.2, a dimer of Fab which itself is a light chain
joined to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2
may be reduced under mild conditions to break the disulfide linkage
in the hinge region thereby converting the (Fab').sub.2 dimer into
an Fab' monomer. The Fab' monomer is essentially an Fab with part
of the hinge region (see, Fundamental Immunology, W. E. Paul, ed.,
Raven Press, N.Y. (1993), for a more detailed description of other
antibody fragments). While various antibody fragments are defined
in terms of the digestion of an intact antibody, one of skill will
appreciate that such Fab' fragments may be synthesized de novo
either chemically or by utilizing recombinant DNA methodology.
Thus, the term antibody, as used herein also includes antibody
fragments either produced by the modification of whole antibodies
or synthesized de novo using recombinant DNA methodologies,
including, but are not limited to, Fab'.sub.2, IgG, IgM, IgA, IgE,
scFv, dAb, nanobodies, unibodies, and diabodies. In various
embodiments preferred antibodies include, but are not limited to
Fab'.sub.2, IgG, IgM, IgA, IgE, and single chain antibodies, more
preferably single chain Fv (scFv) antibodies in which a variable
heavy and a variable light chain are joined together (directly or
through a peptide linker) to form a continuous polypeptide.
[0024] In certain embodiments antibodies and fragments used in the
constructs of the present invention can be bispecific. Bispecific
antibodies or fragments can be of several configurations. For
example, bispecific antibodies may resemble single antibodies (or
antibody fragments) but have two different antigen binding sites
(variable regions). In various embodiments bispecific antibodies
can be produced by chemical techniques (Kranz et al. (1981) Proc.
Natl. Acad. Sci., USA, 78: 5807), by "polydoma" techniques (see,
e.g., U.S. Pat. No. 4,474,893), or by recombinant DNA techniques.
In certain embodiments bispecific antibodies of the present
invention can have binding specificities for at least two different
epitopes at least one of which is a tumor associate antigen. In
various embodiments the antibodies and fragments can also be
heteroantibodies. Heteroantibodies are two or more antibodies, or
antibody binding fragments (e.g., Fab) linked together, each
antibody or fragment having a different specificity.
[0025] An "antigen-binding site" or "binding portion" refers to the
part of an immunoglobulin molecule that participates in antigen
binding. The antigen binding site is formed by amino acid residues
of the N-terminal variable ("V") regions of the heavy ("H") and
light ("L") chains. Three highly divergent stretches within the V
regions of the heavy and light chains are referred to as
"hypervariable regions" which are interposed between more conserved
flanking stretches known as "framework regions" or "FRs". Thus, the
term "FR" refers to amino acid sequences that are naturally found
between and adjacent to hypervariable regions in immunoglobulins.
In an antibody molecule, the three hypervariable regions of a light
chain and the three hypervariable regions of a heavy chain are
disposed relative to each other in three dimensional space to form
an antigen binding "surface". This surface mediates recognition and
binding of the target antigen. The three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity determining regions" or "CDRs" and are
characterized, for example by Kabat et al. Sequences of proteins of
immunological interest, 4th ed. U.S. Dept. Health and Human
Services, Public Health Services, Bethesda, Md. (1987).
[0026] The terms "label" or "detectable label" are used herein to
refer to any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means. Such labels include biotin for staining with
labeled streptavidin conjugate, magnetic beads (e.g.,
DYNABEADS.RTM.), fluorescent dyes (e.g., fluorescein, texas red,
rhodamine, green fluorescent protein, and the like), radiolabels
(e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horse radish
peroxidase, alkaline phosphatase and others commonly used in an
ELISA), and colorimetric labels such as colloidal gold or colored
glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)
beads. Patents teaching the use of such labels include U.S. Pat.
Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149; and 4,366,241. Means of detecting such labels are well
known to those of skill in the art. Thus, for example, radiolabels
may be detected using photographic film or scintillation counters,
fluorescent markers may be detected using a photodetector to detect
emitted light. Enzymatic labels are typically detected by providing
the enzyme with a substrate and detecting the reaction product
produced by the action of the enzyme on the substrate, and
colorimetric labels are detected by simply visualizing the colored
label.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1A-1G show that activation of LXR inhibits LDL uptake
through reduction in LDLR protein expression. FIG. 1A: BODIPY-LDL
binding and uptake in HepG2 cells treated with DMSO or the
synthetic LXR ligands GW3965 (GW) and T0901317 (T), (n=6). FIG. 1B:
Immunoblot analysis of total HepG2 cell lysates. Cells were
pretreated with DMSO or GW (1 .mu.M) for 8 h and subsequently grown
in LPDS, or in sterol depletion medium (LPDS supplemented with 5
.mu.M simvastatin and 100 .mu.M mevalonic acid) containing either
DMSO or GW for an additional 18 h. The precursor (p) and mature (m)
forms of the LDLR are indicated. FIG. 1C: Gene expression in HepG2
cells (n=5) following the indicated treatments was determined by
realtime PCR. FIG. 1D: Immunoblot analysis of total SV589 cell
lysates. Cells were pre-treated with the LXR ligands GW or T (1
.mu.M) for 8 h and subsequently cultured in sterol depletion medium
for an additional 18 h. FIG. 1E: Immortalized MEFs from
Lxr.alpha..beta.(-/-) mice (DKO) and cells stably reconstituted
with mLxr.alpha. were pretreated with GW (1 .mu.M) or
22R-hydroxycholesterol (2.4 .mu.M) for 8 hrs and subsequently
cultured in sterol depletion medium for an additional 18 h. FIG.
1F: Immunoblot analysis of total SV589 cell lysates. Cell were
grown in 10% FBS and infected with adenovirus expressing GFP or
Sult2b1 for 24 h and then cultured in sterol depletion medium for
an additional 18 h. FIG. 1G: Immunofluorescence images of
HepG2-LDLR-GFP cells treated with DMSO, GW and T (1 .mu.M) for 72
hrs. *p<0.001. Error bars represent the mean.+-.SD.
[0028] FIGS. 2A-2K show the LXR target gene Mylip is a regulator of
LDLR protein expression. FIG. 2A: Induction of Mylip mRNA
expression by GW or T (1 .mu.M) in SV589 cells, HepG2 cells and in
primary mouse astrocytes. FIG. 2B: LXR-dependent regulation of
Mylip in MEFs and primary mouse hepatocytes following treatment
with GW, T (both at 1 .mu.M) or 22R-hydroxycholesterol (2.5 .mu.M).
FIG. 2C: Regulation of Mylip by LXR in vivo. Mice were gavaged with
20 mg/kg GW twice daily for three days and gene expression was
analyzed by realtime PCR (n=6 mice/group). FIG. 2D: Dose-dependent
reduction of LDLR-GFP protein in HEK293 cells co-transfected with
Mylip and LDLR-GFP expression plasmids. Arrow indicates the Mylip
protein. FIG. 2E: Immunofluorescence images of HEK293 cells
co-transfected with LDLR-GFP and either wildtype or enzymatic
mutant human and mouse Mylip. FIG. 2F: Immunoblot analysis of total
293 cell lysates co-transfected with Mylip constructs and the
indicated GFP fusion proteins. Endogenous TFRC and MRLC are also
shown. FIG. 2G: Primary hepatocytes were isolated and infected with
Ad-GFP or Ad-Mylip for 24 h followed by culture in sterol depletion
medium for an additional 18 h. FIG. 2H: BODIPY-LDL binding and
uptake in DKO-mLxr.alpha. cells following infection with Ad-LacZ or
Ad-Mylip (n=6); FIG. 2I: DKO-mLxr.alpha. cells were infected with
control (shLamin) or two independent adenoviral Mylip shRNA
constructs for 24 h and subsequently cultured for 18 h in sterol
depletion medium. FIG. 2J: BODIPY-LDL binding and uptake in
DKO-mLxr.alpha. cells following infection with Ad-shLAMIN,
Ad-shMylip1, or Ad-shMylip2 (n=4). FIG. 2K: DKO-mLxr.alpha. cells
were infected with the indicated adenovirus for 24 h. Subsequently,
cells were pre-treated for 8 h with DMSO or GW (1 .mu.M) followed
by an additional 18 h in sterol depletion medium. A representative
of 4 independent experiments is shown *p<0.05, **p<0.01,
***p<0.001.
[0029] FIGS. 3A-3G, show that Mylip reduces LDLR protein expression
through ubiquitination of conserved residues in its cytoplasmic
domain. FIG. 3A: 24 h following infection with Ad-LacZ or Ad-Mylip
HepG2-LDLR-GFP cells were metabolically labelled with
[.sup.35S]Methionine and [.sup.35S]Cysteine for 30 minutes. Samples
were immunopreciptiated at the indicated time points following
labeling. FIG. 3B: HEK293 cells were co-transfected with LDLR-GFP,
Mylip and HA-Ubiquitin expression plasmids. Subsequently, samples
were immunoblotted as indicated. FIG. 3C: Immunoblot analysis of
total HEK293 cell lysates 48 h after co-transfection with Mylip and
LDLR expression plasmids. FIG. 3D: Evolutionary conservation of the
LDLR intracellular domain. Residues that serve as potential
ubiquitination sites are indicated. H. sapiens (SEQ ID NO:1), P.
troglodytes (SEQ ID NO:2), R. norvegicus (SEQ ID NO:3), M. musculus
(SEQ ID NO:4), O. cuniculus (SEQ ID NO:5); FIGS. 3E and 3F:
Immunoblot analysis of HEK293 total cell lysates co-transfected
with control or Mylip expression plasmids along with the indicated
mutated LDLR constructs. Numbering in the LDLR constructs refers to
FIG. 1D. FIG. 3G: HEK293 cells were co-transfected with LDLR, Mylip
and HA-Ubiquitin expression plasmids. Subsequently, cells were
treated with vehicle or 25 .mu.M MG132 for 6 h.
[0030] FIGS. 4A-4G show that expression of Mylip reduces LDLR
expression and elevates plasma cholesterol and LDL levels in vivo.
FIG. 4A: Analysis of plasma 6 days after transduction of C57BL/6
mice with Ad-LacZ or Ad-Mylip. FFA, free fatty acids (n=8
mice/group). FIGS. 4B and 4C: Cholesterol and triglyceride content
of fractionated plasma from mice infected with Ad-LacZ or Ad-Mylip.
FIG. 4D: Immunodetection of ApoB100, and ApoB48 in fractionated
plasma of Ad-LacZ and Ad-Mylip infected mice. FIG. 4E: Gene
expression in livers of Ad-LacZ and Ad-Mylip infected mice. FIG.
4F: Immunoblot analysis of total liver lysates. FIG. 4G:
Cholesterol content of fractionated plasma from Ldlr(-/-) mice
infected with Ad-LacZ, or Ad-Mylip. ***p<0.001.
[0031] FIG. 5 shows the effect of LXR activation on LDLR mRNA
expression in SV589 cells. Gene expression in SV589 cells cultured
in 10% FBS or sterol depletion medium was determined 24 h after
treatment with LXR agonists GW or T (1 .mu.M).
[0032] FIGS. 6A-6E illustrate the identification of the Mylip gene
as a direct target for LXR regulation. FIG. 6A: Mylip expression in
RAW264.7 macrophage cells following treatment with the LXR ligand
GW (1 .mu.M), the RXR ligand LG268 (50 nM), and/or cycloheximide
(CHX, 10 .mu.g/mL). FIG. 6B: Mylip expression in
thioglycolate-elicited peritoneal macrophages following 24 h
treatment with DMSO, GW (1 .mu.M), or 25-hydroxycholesterol (2.5
.mu.M). FIG. 6C: Sequence of the LXRE in the mouse Mylip proximal
promoter. Mylip WT DR4 sense strand (SEQ ID NO:6), Mylip WT DR4
antisense strand (SEQ ID NO:7), Mylip MUT DR4 sense strand (SEQ ID
NO:8), Mylip MUT DR4 antisense strand (SEQ ID NO:9). FIG. 6D:
HEK293 cells were transfected with LXR and RXR expression vectors
along with the indicated promoter constructs and treated for 48 h
with GW (1 .mu.M) and LG268 (50 nM). FIG. 6E: EMSA analysis of
LXR/RXR binding to radiolabeled Mylip LXRE.
[0033] FIG. 7 shows that expression of Mylip inhibits plasma
membrane expression of the LDLR. Low magnification
immunofluorescence images of HEK293 cells co-transfected with
LDLR-GFP and Mylip constructs
[0034] FIGS. 8A and 8B show the effect of Mylip knockdown on LDLR
mRNA and protein expression in MEFs. FIG. 8A: Gene expression in
DKO-Lxr MEFs following transduction with the indicated adenoviral
shRNA constructs determined by realtime PCR. FIG. 8B: Effect of
Mylip knockdown on LDLR is comparable to sterol depletion.
Immunoblot analysis of total DKO-Lxr cell lysates. Where indicated
cells were infected with Ad-shLAMIN or AdshMylip for 24 h.
Subsequently, cells were culture in 10% LPDS, or sterol depletion
medium for an additional 18 h.
[0035] FIGS. 9A and 9B show the effect of Mylip on LDLR protein
expression. FIG. 9A: The NPXY endocytosis motif is not required for
Mylip inhibition of LDLR expression. Immunoblot analysis of total
HEK293 cell lysates following co-transfection with plasmids
encoding Mylip and/or LDLR-GFP or LDLR(Y18A)-GFP. Figure B:
Mutation of 128 in the LDLR cytoplasmic domain does not inhibit
Mylip action on the LDLR. Immunoblot analysis of total HEK293 cell
lysates following co-transfection with plasmids encoding Mylip and
LDLR various LDLR mutants as indicated.
[0036] FIGS. 10A-10E show that adenoviral expression of Mylip
regulates plasma cholesterol levels in vivo. FIG. 10A: Analysis of
plasma from Ad-LacZ and Ad-Mylip infected C57BL/6 mice. FFA, free
fatty acids (n=8 mice/group). FIG. 10B: Cholesterol content of
fractionated plasma from mice infected with Ad-LacZ, or Ad-Mylip.
FIG. 10C: Gene expression in livers of AdLacZ, or Ad-Mylip infected
mice determined by realtime PCR.
[0037] FIG. 10D: Analysis of plasma from Ad-LacZ and Ad-Mylip
infected C57BL/6 Ldlr(-/-) mice (n=5 mice/group). FIG. 10E:
Triglyceride content of fractionated plasma from C57BL/6 Ldlr(-/-)
mice) infected with Ad-LacZ, or Ad-Mylip (n=5 mice/group).
[0038] FIGS. 11A and 11B illustrate knockdown of Mylip expression
by multiple shRNA sequences. Immunoblot analysis of total HEK293
cell lysates following co-transfection with pENTR-U6-shLacZ
(control), or with four independent shRNA constructs targeting
mouse Mylip and (FIG. 11A) non-tagged mutated mMylip (C387A), and
(FIG. 11B) pEGFP-N-3-wt mMylip.
[0039] FIGS. 12A and 12B show data regarding the generation and
validation of homozygous knockout Idol ES cells. FIG. 12A: Realtime
PCR analysis of mRNA expression in WT and Idol-/- ES cells. FIG.
12B: Immunoblot analysis of LDLR protein expression in WT and
Idol-/- ES cells treated with vehicle or LXR agonist.
[0040] FIG. 13 illustrates the domain structure of Idol (mylip).
The protein contains an N-terminal FERM protein-protein interaction
domain and a C-terminal RING domain characteristic of E3 ubiquitin
ligases.
[0041] FIG. 14 shows the results of an analysis of Idol functional
domains. HEK293 cells were transiently transfected with LDLR and/or
WT or mutant Idol constructs as indicated. LDLR expression was
determined by immunoblotting. C387A is a point mutation that
inactivates the RING domain.
[0042] FIG. 15 shows an alignment of cytoplasmic tails of candidate
membrane protein targets of Idol. Ubiquitination sites in the LDLR
are shown in the areas labeled with asterisks. The NPVY
internalization signal shown in the box labeled with
.tangle-solidup. highlighted blue. Other regions of sequence
similarity are in the boxes labeled with .DELTA.. LDLR (SEQ ID
NO:10), VLDLR (SEQ ID NO:11), apoER2 (SEQ ID NO:12), LRP1B (SEQ ID
NO:13), LRP1 (SEQ ID NO:14), EGFR (SEQ ID NO:15).
[0043] FIG. 16 shows data illustrating the validation of
liver-specific Idol transgenic mice. Expression of human transgene
specific Idol expression in livers of Albumin-Idol Tg mice
determined by realtime PCR.
[0044] FIG. 17 illustrates a knockout strategy for the mouse Idol
locus. The targeting vector is conditional-ready and the null
allele can be converted to a floxed allele by crossing with
FLIP-expressing mice. This will yield an allele with exon2
[0045] FIG. 18 shows data from the PCR validation of Idol null
mice. Tail DNA was analyzed by PCR for the presence of WT and null
Idol alleles.
DETAILED DESCRIPTION
[0046] In various embodiments this invention pertains to a novel
hepatic target for treatment of hypercholesterolemia. In
particular, in certain embodiments, this invention pertains to the
discovery that myosin light chain interacting protein (Mylip) (also
known as Inducible Degrader of the LDLR or IDOL) is a regulator of
cholesterol homeostasis. Mylip is a transcriptional target of the
Liver X Receptors (LXR5) in different murine and human cells types
and tissues including liver. Mylip is a highly conserved ERM-like
protein that contains a C-terminal RING domain. Mylip is thought to
function as an E3-ubiquitin ligase, but its protein targets and
physiological roles are unknown.
[0047] Our research identified Mylip as an in vivo regulator of
hepatic levels of the LDLR, independent of the cholesterol
biosynthetic pathway that is targeted by statins.
Adenoviral-mediated hepatic expression of Mylip results in severely
diminished LDLR levels and increased total cholesterol and LDL
levels. Similarly, Mylip reduces LDLR in vitro. Importantly, this
activity depends on an intact RING (the E3-ubiquitin-ligase) domain
suggesting that the ability of Mylip to reduce the LDLR depends on
its enzymatic activity. Thus, genetic methods or chemicals that can
inhibit the expression and/or activity of Mylip in the liver can
form the basis for a new class of drugs for treating
hypercholesterolemia and cardiac disease.
[0048] Accordingly, in certain embodiments, methods are provided
for inhibiting LDL receptor degradation and/or promoting LDL uptake
in a mammal, and/or one mitigating or more symptoms of
hypercholesterolemia in a mammal. The methods typically involve
administering to the mammal an agent that inhibits the expression
and/or activity of Mylip. Methods are also provided of screening
for such agents.
I. Assays for Inhibitors of Myosin Light Chain Interacting Protein
(Mylip)
[0049] As indicated above, it was a surprising discovery that
inhibition of Mylip expression or activity can inhibit LDL receptor
degradation and/or promote LDL uptake in a mammal, and/or mitigate
one or more symptoms of hypercholesterolemia. Mylip thus provides a
target to screen for agents that inhibit LDL receptor degradation
and/or promote LDL uptake in a mammal, and/or mitigate one or more
symptoms of. In various embodiments the methods can involve
contacting a cell, a tissue, an organism with one or more test
agents and detecting (resulting changes in) the expression level
and/or activity level of. Inhibition of Mylip expression tore of an
aging process.
[0050] It is noted that when screening for Mylip inhibitors, a
positive assay result need not indicate the particular test agent
is a good pharmaceutical. Rather a positive result can simply
indicate that the test agent can be used to inhibit Mylip activity
and/or can also serve as a lead compound in the development of
other modulators.
[0051] Using the methods described herein, test agents can readily
be screened for the ability to inhibit Mylip expression and/or
activity.
[0052] Activity-Based Assays.
[0053] For example, inhibition of Mylip expression and/or activity
can readily be determined by placing one or more reporter genes
under control of a promoter system whose regulation is controlled
by Mylip or the Mylip promoter. Cell(s) containing such constructs
are contacted with test agents, and a reduction in the reporter
indicates that the test agent(s) inhibit Mylip expression or
activity.
[0054] We have developed the following cell based assay
systems/strategies for high throughput screening of Mylip/Idol
inhibitors (see, e.g., Example 1). While specific examples of cell
types, vectors, transfection strategies, tags are given, it will be
recognized that these are illustrative and not limiting. Using the
teachings provided herein, other cells, constructs, labels, and
assay formats will be available to one of skill in the art.
[0055] The assays described below can also be employed in
combination, with one serving as the primary screen and the others
as secondary screens. In certain embodiments two of these assays
could also be combined in the same reporter cell line (e.g. a
single cell line that expresses both LDLR (1) and Mylip (2)
reporters):
[0056] Cell Based Assay for Inhibitors of Mylip Action on the
LDLR.
[0057] In certain embodiments cell based assays can be used to
screen test agents (potential inhibitors) for inhibition of Mylip
action on the LDL receptor (LDLR).
[0058] For example, a reporter cell line (e.g. HEK293, HepG2 cells)
can be transfected (stably, transiently, virally etc.) with a
tagged version of the LDLR, expression of which can be readily
assayed by HTS methods. For example LDLR-luciferase fusion protein
expression can be assayed by luciferase activity. Alternatively,
LDLR-GFP fusion protein can be assayed by high content imaging.
Other variations on this idea are possible.
[0059] A second component of the assay is to co-transfect (either
stably or transiently or by adenoviral transduction) the reporter
cells with active Mylip/Idol. In basal state, this results in very
little or no signal from the LDLR-fusion protein because it is
degraded by Mylip (see, e.g., FIG. 2E). HTS screening is used to
identify small molecules, RNAs, genes etc that block Mylip action
and thereby increase expression of LDLR-fusion reporter.
[0060] Cell Based Assay for Inhibitors of Mylip
Auto-Degradation.
[0061] Another approach uses cell-based assays for inhibitors of
Mylip auto-degradation. These assays take advantage of our
discovery that Mylip E3 ligase activity leads to auto-degradation
of Mylip protein. Thus, Mylip inhibitors can be identified by
screening for compounds, genes, RNAs etc that stabilize Mylip
protein expression (see FIGS. 2F and 2I). A reporter cell line
(e.g. HEK293 cells, HepG2 cells) is transfected (stably or
transiently, virally etc) with a tagged version of Mylip,
expression of which can be readily assayed by HTS methods. For
example, Mylip-luciferase fusion protein expression can be assayed
by luciferase activity. Alternatively, Mylip-GFP fusion protein
expression can be assayed by high content imaging. Other variations
on this idea are possible. In the basal state, reporter cells
express very little signal from the Mylip-luciferase (or other)
fusion protein because Mylip catalyzes its own degradation and is
very unstable. HTS screening is used to identify small molecules,
RNAs, genes etc that block Mylip autodegradation thereby increase
expression of Mylip-fusion reporter.
[0062] Cell Based Assay for Inhibitors of Mylip Action on LDL
Uptake.
[0063] A reporter cell line (e.g. HEK293, HepG2 cells) expressing
the LDLR (either endogenously or through transfection) can be
cotransfected with Mylip expression vector. Uptake of labeled LDL
(e.g., BODIPY-LDL) is determined (e.g., by high content imaging).
In basal state, this results in very little or no labeled LDL
uptake because the LDLR is degraded by Mylip. HTS screening is used
to identify small molecules, RNAs, genes etc that block Mylip
action and thereby increase BODIPY-LDL uptake (see, e.g., FIG.
2J).
[0064] Cell- and Animal-Based Inhibitor Assays.
[0065] In certain embodiments wildtype (WT) and Idol-/- cells or
animals are screened for their response to candidate small molecule
modulators (e.g., inhibitors) of Idol expression and/or activity.
The effect of Idol-specific small molecules is lost or reduced in
the Idol-/- cells, tissues, and/or animals. This screening method
will be used, for example, in conjunction with the cell-based
reporter screens described above.
[0066] Similarly animals, tissues, and/or cells overexpressing idol
can be screened for the activity of candidate inhibitors using, for
example, wildtype cells, and/or tissues, and/or animals as a
control.
[0067] Nucleic-Acid Based Assays.
[0068] Using the known nucleic acid sequences for Mylip, copy
number and/or, Mylip expression level, can be directly measured
according to a number of different methods as described below. In
particular, expression levels of a gene can be altered by changes
in the copy number of the gene, and/or by changes in the
transcription of the gene product (i.e. transcription of mRNA),
and/or by changes in translation of the gene product (i.e.
translation of the protein), and/or by post-translational
modification(s) (e.g. protein folding, glycosylation, etc.). Thus
useful assays of this invention include assaying for copy number,
level of transcribed mRNA, level of translated protein, activity of
translated protein, etc. Examples of such approaches are described
below.
[0069] 1) Target Molecules.
[0070] Changes in expression level can be detected by measuring
changes in mRNA and/or a nucleic acid derived from the mRNA (e.g.
reverse-transcribed cDNA, etc.). In order to measure the Mylip
expression level it is desirable to provide a nucleic acid sample
for such analysis. In certain preferred embodiments the nucleic
acid is found in or derived from a biological sample. The term
"biological sample", as used herein, refers to a sample obtained
from an organism or from components (e.g., cells) of an organism,
or from cells in culture. The sample may be of any biological
tissue or fluid. Biological samples may also include organs or
sections of tissues such as frozen sections taken for histological
purposes.
[0071] The nucleic acid (e.g., mRNA nucleic acid derived from mRNA)
is, in certain preferred embodiments, isolated from the sample
according to any of a number of methods well known to those of
skill in the art. Methods of isolating mRNA are well known to those
of skill in the art. For example, methods of isolation and
purification of nucleic acids are described in detail in by Tijssen
ed., (1993) Chapter 3 of Laboratory Techniques in Biochemistry and
Molecular Biology: Hybridization With Nucleic Acid Probes, Part L
Theory and Nucleic Acid Preparation, Elsevier, N.Y. and Tijssen
ed.
[0072] In a certain embodiments, the "total" nucleic acid is
isolated from a given sample using, for example, an acid
guanidinium-phenol-chloroform extraction method and polyA+ mRNA is
isolated by oligo dT column chromatography or by using (dT)n
magnetic beads (see, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor
Laboratory, (1989), or Current Protocols in Molecular Biology, F.
Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New
York (1987)).
[0073] Frequently it is desirable to amplify the nucleic acid
sample prior to assaying for expression level. Methods of
amplifying nucleic acids are well known to those of skill in the
art and include, but are not limited to polymerase chain reaction
(PCR, see. e.g, Innis, et al., (1990) PCR Protocols. A guide to
Methods and Application. Academic Press, Inc. San Diego,), ligase
chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560,
Landegren et al. (1988) Science 241: 1077, and Barringer et al.
(1990) Gene 89: 117, transcription amplification (Kwoh et al.
(1989) Proc. Natl. Acad. Sci. USA.sub.--86: 1173), self-sustained
sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci.
USA 87: 1874), dot PCR, and linker adapter PCR, etc.).
[0074] In one illustrative embodiment, where it is desired to
quantify the transcription level (and thereby expression) of Mylip
in a sample, the nucleic acid sample is one in which the
concentration of the Mylip mRNA transcript(s), or the concentration
of the nucleic acids derived from the Mylip mRNA transcript(s), is
proportional to the transcription level (and therefore expression
level) of that gene. Similarly, it is preferred that the
hybridization signal intensity be proportional to the amount of
hybridized nucleic acid. While it is preferred that the
proportionality be relatively strict (e.g., a doubling in
transcription rate results in a doubling in mRNA transcript in the
sample nucleic acid pool and a doubling in hybridization signal),
one of skill will appreciate that the proportionality can be more
relaxed and even non-linear. Thus, for example, an assay where a 5
fold difference in concentration of the target mRNA results in a 3
to 6 fold difference in hybridization intensity is sufficient for
most purposes.
[0075] Where more precise quantification is required appropriate
controls can be run to correct for variations introduced in sample
preparation and hybridization as described herein. In addition,
serial dilutions of "standard" target nucleic acids (e.g., mRNAs)
can be used to prepare calibration curves according to methods well
known to those of skill in the art. Of course, where simple
detection of the presence or absence of a transcript or large
differences of changes in nucleic acid concentration is desired, no
elaborate control or calibration is required.
[0076] In one simple embodiment, the Mylip-containing nucleic acid
sample is the total mRNA or a total cDNA isolated and/or otherwise
derived from a biological sample. The nucleic acid may be isolated
from the sample according to any of a number of methods well known
to those of skill in the art as indicated above.
[0077] 2) Hybridization-Based Assays.
[0078] Using known Mylip sequences detecting and/or quantifying the
Mylip transcript(s) can be routinely accomplished using nucleic
acid hybridization techniques (see, e.g., Sambrook et al. supra).
For example, one method for evaluating the presence, absence, or
quantity of Mylip reverse-transcribed cDNA involves a "Southern
Blot". In a Southern Blot, the DNA (e.g., reverse-transcribed Mylip
mRNA), typically fragmented and separated on an electrophoretic
gel, is hybridized to a probe specific for Mylip (or to a mutant
thereof). Comparison of the intensity of the hybridization signal
from the Mylip probe with a "control" probe (e.g. a probe for a
"housekeeping gene) provides an estimate of the relative expression
level of the target nucleic acid.
[0079] Alternatively, the Mylip mRNA can be directly quantified in
a Northern blot. In brief, the mRNA is isolated from a given cell
sample using, for example, an acid guanidinium-phenol-chloroform
extraction method. The mRNA is then electrophoresed to separate the
mRNA species and the mRNA is transferred from the gel to a
nitrocellulose membrane. As with the Southern blots, labeled probes
are used to identify and/or quantify the target Mylip mRNA.
Appropriate controls (e.g. probes to housekeeping genes) provide a
reference for evaluating relative expression level.
[0080] An alternative means for determining the Mylip expression
level is in situ hybridization. In situ hybridization assays are
well known (e.g., Angerer (1987) Meth. Enzymol 152: 649).
Generally, in situ hybridization comprises the following major
steps: (1) fixation of tissue or biological structure to be
analyzed; (2) prehybridization treatment of the biological
structure to increase accessibility of target DNA, and to reduce
nonspecific binding; (3) hybridization of the mixture of nucleic
acids to the nucleic acid in the biological structure or tissue;
(4) post-hybridization washes to remove nucleic acid fragments not
bound in the hybridization and (5) detection of the hybridized
nucleic acid fragments. The reagent used in each of these steps and
the conditions for use vary depending on the particular
application.
[0081] In some applications it is necessary to block the
hybridization capacity of repetitive sequences. Thus, in some
embodiments, tRNA, human genomic DNA, or Cot-1 DNA is used to block
non-specific hybridization.
[0082] 3) Amplification-Based Assays.
[0083] In another embodiment, amplification-based assays can be
used to measure Mylip expression (transcription) level. In such
amplification-based assays, the target nucleic acid sequences
(i.e., Mylip) act as template(s) in amplification reaction(s) (e.g.
Polymerase Chain Reaction (PCR) or reverse-transcription PCR, or
quantitative PCR (e.g., quantitative RT-PCR). In a quantitative
amplification, the amount of amplification product will be
proportional to the amount of template (e.g., Mylip mRNA) in the
original sample. Comparison to appropriate (e.g. healthy tissue or
cells unexposed to the test agent) controls provides a measure of
the Mylip transcript level.
[0084] Methods of "quantitative" amplification are well known to
those of skill in the art. For example, quantitative PCR involves
simultaneously co-amplifying a known quantity of a control sequence
using the same primers. This provides an internal standard that may
be used to calibrate the PCR reaction. Detailed protocols for
quantitative PCR are provided in Innis et al. (1990) PCR Protocols,
A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
One approach, for example, involves simultaneously co-amplifying a
known quantity of a control sequence using the same primers as
those used to amplify the target. This provides an internal
standard that may be used to calibrate the PCR reaction.
[0085] One typical internal standard is a synthetic AW106 cRNA. The
AW106 cRNA is combined with RNA isolated from the sample according
to standard techniques known to those of skill in the art. The RNA
is then reverse transcribed using a reverse transcriptase to
provide copy DNA. The cDNA sequences are then amplified (e.g., by
PCR) using labeled primers. The amplification products are
separated, typically by electrophoresis, and the amount of labeled
nucleic acid (proportional to the amount of amplified product) is
determined. The amount of mRNA in the sample is then calculated by
comparison with the signal produced by the known AW106 RNA
standard. Detailed protocols for quantitative PCR are provided in
PCR Protocols, A Guide to Methods and Applications, Innis et al.
(1990) Academic Press, Inc. N.Y. The known nucleic acid sequence(s)
for MYLIP are sufficient to enable one of skill to routinely select
primers to amplify any portion of the gene.
[0086] 4) Hybridization Formats and Optimization of Hybridization
Conditions.
[0087] a) Array-Based Hybridization Formats.
[0088] In one embodiment, the methods of this invention can be
utilized in array-based hybridization formats. Arrays are a
multiplicity of different "probe" or "target" nucleic acids (or
other compounds) attached to one or more surfaces (e.g., solid,
membrane, or gel). In a certain embodiments, the multiplicity of
nucleic acids (or other moieties) is attached to a single
contiguous surface or to a multiplicity of surfaces juxtaposed to
each other.
[0089] In an array format a large number of different hybridization
reactions can be run essentially "in parallel." This provides
rapid, essentially simultaneous, evaluation of a number of
hybridizations in a single "experiment". Methods of performing
hybridization reactions in array based formats are well known to
those of skill in the art (see, e.g., Pastinen (1997) Genome Res.
7: 606-614; Jackson (1996) Nature Biotechnology 14:1685; Chee
(1995) Science 274: 610; WO 96/17958, Pinkel et al. (1998) Nature
Genetics 20: 207-211).
[0090] Arrays, particularly nucleic acid arrays can be produced
according to a wide variety of methods well known to those of skill
in the art. For example, in a simple embodiment, "low density"
arrays can simply be produced by spotting (e.g. by hand using a
pipette) different nucleic acids at different locations on a solid
support (e.g. a glass surface, a membrane, etc.).
[0091] This simple spotting, approach has been automated to produce
high density spotted arrays (see, e.g., U.S. Pat. No. 5,807,522).
This patent describes the use of an automated system that taps a
microcapillary against a surface to deposit a small volume of a
biological sample. The process is repeated to generate high-density
arrays.
[0092] Arrays can also be produced using oligonucleotide synthesis
technology. Thus, for example, U.S. Pat. No. 5,143,854 and PCT
Patent Publication Nos. WO 90/15070 and 92/10092 teach the use of
light-directed combinatorial synthesis of high density
oligonucleotide arrays. Synthesis of high-density arrays is also
described in U.S. Pat. Nos. 5,744,305, 5,800,992 and 5,445,934.
[0093] b) Other Hybridization Formats.
[0094] As indicated above a variety of nucleic acid hybridization
formats are known to those skilled in the art. For example, common
formats include sandwich assays and competition or displacement
assays. Such assay formats are generally described in Hames and
Higgins (1985) Nucleic Acid Hybridization, A Practical Approach,
IRL Press; Gall and Pardue (1969) Proc. Natl. Acad. Sci. USA 63:
378-383; and John et al. (1969) Nature 223: 582-587.
[0095] Sandwich assays are commercially useful hybridization assays
for detecting or isolating nucleic acid sequences. Such assays
utilize a "capture" nucleic acid covalently immobilized to a solid
support and a labeled "signal" nucleic acid in solution. The sample
will provide the target nucleic acid. The "capture" nucleic acid
and "signal" nucleic acid probe hybridize with the target nucleic
acid to form a "sandwich" hybridization complex. To be most
effective, the signal nucleic acid should not hybridize with the
capture nucleic acid.
[0096] Typically labeled signal nucleic acids are used to detect
hybridization. Complementary nucleic acids or signal nucleic acids
may be labeled by any one of several methods typically used to
detect the presence of hybridized polynucleotides. The most common
method of detection is the use of autoradiography with .sup.3H,
.sup.125I, .sup.35S, .sup.14C, or .sup.32P-labelled probes or the
like. Other labels include ligands that bind to labeled antibodies,
fluorophores, chemi-luminescent agents, enzymes, and antibodies
that can serve as specific binding pair members for a labeled
ligand.
[0097] Detection of a hybridization complex may require the binding
of a signal generating complex to a duplex of target and probe
polynucleotides or nucleic acids. Typically, such binding occurs
through ligand and anti-ligand interactions as between a
ligand-conjugated probe and an anti-ligand conjugated with a
signal.
[0098] The sensitivity of the hybridization assays may be enhanced
through use of a nucleic acid amplification system that multiplies
the target nucleic acid being detected. Examples of such systems
include the polymerase chain reaction (PCR) system and the ligase
chain reaction (LCR) system. Other methods recently described in
the art are the nucleic acid sequence based amplification (NASBAO,
Cangene, Mississauga, Ontario) and Q Beta Replicase systems.
[0099] c) Optimization of Hybridization Conditions.
[0100] Nucleic acid hybridization simply involves providing a
denatured probe and target nucleic acid under conditions where the
probe and its complementary target can form stable hybrid duplexes
through complementary base pairing. The nucleic acids that do not
form hybrid duplexes are then washed away leaving the hybridized
nucleic acids to be detected, typically through detection of an
attached detectable label. It is generally recognized that nucleic
acids are denatured by increasing the temperature or decreasing the
salt concentration of the buffer containing the nucleic acids, or
in the addition of chemical agents, or the raising of the pH. Under
low stringency conditions (e.g., low temperature and/or high salt
and/or high target concentration) hybrid duplexes (e.g., DNA:DNA,
RNA:RNA, or RNA:DNA) will form even where the annealed sequences
are not perfectly complementary. Thus specificity of hybridization
is reduced at lower stringency. Conversely, at higher stringency
(e.g., higher temperature or lower salt) successful hybridization
requires fewer mismatches.
[0101] One of skill in the art will appreciate that hybridization
conditions may be selected to provide any degree of stringency. In
a preferred embodiment, hybridization is performed at low
stringency to ensure hybridization and then subsequent washes are
performed at higher stringency to eliminate mismatched hybrid
duplexes. Successive washes may be performed at increasingly higher
stringency (e.g., down to as low as 0.25.times.SSPE at 37.degree.
C. to 70.degree. C.) until a desired level of hybridization
specificity is obtained. Stringency can also be increased by
addition of agents such as formamide. Hybridization specificity may
be evaluated by comparison of hybridization to the test probes with
hybridization to the various controls that can be present.
[0102] In general, there is a tradeoff between hybridization
specificity (stringency) and signal intensity. Thus, in a preferred
embodiment, the wash is performed at the highest stringency that
produces consistent results and that provides a signal intensity
greater than approximately 10% of the background intensity. Thus,
in a preferred embodiment, the hybridized array may be washed at
successively higher stringency solutions and read between each
wash. Analysis of the data sets thus produced will reveal a wash
stringency above which the hybridization pattern is not appreciably
altered and which provides adequate signal for the particular
probes of interest.
[0103] In a preferred embodiment, background signal is reduced by
the use of a blocking reagent (e.g., tRNA, sperm DNA, cot-1 DNA,
etc.) during the hybridization to reduce non-specific binding. The
use of blocking agents in hybridization is well known to those of
skill in the art (see, e.g., Chapter 8 in P. Tijssen, supra.).
[0104] Methods of optimizing hybridization conditions are well
known to those of skill in the art (see, e.g., Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular Biology, Vol.
24: Hybridization With Nucleic Acid Probes, Elsevier, N.Y.).
[0105] Optimal conditions are also a function of the sensitivity of
label (e.g., fluorescence) detection for different combinations of
substrate type, fluorochrome, excitation and emission bands, spot
size and the like. Low fluorescence background surfaces can be used
(see, e.g., Chu (1992) Electrophoresis 13:105-114). The sensitivity
for detection of spots ("target elements") of various diameters on
the candidate surfaces can be readily determined by, e.g., spotting
a dilution series of fluorescently end labeled DNA fragments. These
spots are then imaged using conventional fluorescence microscopy.
The sensitivity, linearity, and dynamic range achievable from the
various combinations of fluorochrome and solid surfaces (e.g.,
glass, fused silica, etc.) can thus be determined. Serial dilutions
of pairs of fluorochrome in known relative proportions can also be
analyzed. This determines the accuracy with which fluorescence
ratio measurements reflect actual fluorochrome ratios over the
dynamic range permitted by the detectors and fluorescence of the
substrate upon which the probe has been fixed.
[0106] d) Labeling and Detection of Nucleic Acids.
[0107] The probes used herein for detection of Mylip expression
levels can be full length or less than the full length of the Mylip
or mutants thereof. Shorter probes are empirically tested for
specificity. Preferred probes are sufficiently long so as to
specifically hybridize with the Mylip target nucleic acid(s) under
stringent conditions. The preferred size range is from about 10,
15, or 20 bases to the length of the Mylip mRNA, more preferably
from about 30 bases to the length of the Mylip mRNA, and most
preferably from about 40 bases to the length of the Mylip mRNA. The
probes are typically labeled, with a detectable label as described
above.
[0108] Polypeptide-Based Assays.
[0109] The effect of a test agent on MYLIP expression can also be
determined by determining the effect of that test agent on
translated Mylip protein.
[0110] The polypeptide(s) encoded by the Mylip gene can be detected
and quantified by any of a number of methods well known to those of
skill in the art. These may include analytic biochemical methods
such as electrophoresis, capillary electrophoresis, high
performance liquid chromatography (HPLC), thin layer chromatography
(TLC), hyperdiffusion chromatography, mass spectroscopy, and the
like, or various immunological methods such as fluid or gel
precipitin reactions, immunodiffusion (single or double),
immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked
immunosorbent assays (ELISAs), immunofluorescent assays, western
blotting, and the like.
[0111] In one preferred embodiment, the Mylip polypeptide(s) are
detected/quantified in an electrophoretic protein separation (e.g.
a 1- or 2-dimensional electrophoresis). Means of detecting proteins
using electrophoretic techniques are well known to those of skill
in the art (see generally, R. Scopes (1982) Protein Purification,
Springer-Verlag, N.Y.; Deutscher, (1990) Methods in Enzymology Vol.
182: Guide to Protein Purification, Academic Press, Inc.,
N.Y.).
[0112] In another preferred embodiment, Western blot (immunoblot)
analysis is used to detect and quantify the presence of
polypeptide(s) of this invention in the sample. This technique
generally comprises separating sample proteins by gel
electrophoresis on the basis of molecular weight, transferring the
separated proteins to a suitable solid support, (such as a
nitrocellulose filter, a nylon filter, or derivatized nylon
filter), and incubating the sample with the antibodies that
specifically bind the target polypeptide(s).
[0113] The antibodies specifically bind to the target
polypeptide(s) and may be directly labeled or alternatively may be
subsequently detected using labeled antibodies (e.g., labeled sheep
anti-mouse antibodies) that specifically bind to a domain of the
antibody.
[0114] In preferred embodiments, the Mylip polypeptide(s) are
detected using an immunoassay. As used herein, an immunoassay is an
assay that utilizes an antibody to specifically bind to the analyte
(e.g., the target polypeptide(s)). The immunoassay is thus
characterized by detection of specific binding of a polypeptide of
this invention to an antibody as opposed to the use of other
physical or chemical properties to isolate, target, and quantify
the analyte.
[0115] Any of a number of well recognized immunological binding
assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288;
and 4,837,168) are well suited to detection or quantification of
the polypeptide(s) identified herein. For a review of the general
immunoassays, see also Asai (1993) Methods in Cell Biology Volume
37: Antibodies in Cell Biology, Academic Press, Inc. New York;
Stites & Terr (1991) Basic and Clinical Immunology 7th
Edition.
[0116] Immunological binding assays (or immunoassays) typically
utilize a "capture agent" to specifically bind to and often
immobilize the analyte (Mylip polypeptide). In preferred
embodiments, the capture agent is an antibody.
[0117] Immunoassays also often utilize a labeling agent to
specifically bind to and label the binding complex formed by the
capture agent and the analyte. The labeling agent may itself be one
of the moieties comprising the antibody/analyte complex. Thus, the
labeling agent may be a labeled polypeptide or a labeled antibody
that specifically recognizes the already bound target polypeptide.
Alternatively, the labeling agent may be a third moiety, such as
another antibody, that specifically binds to the capture
agent/polypeptide complex.
[0118] Other proteins capable of specifically binding
immunoglobulin constant regions, such as protein A or protein G may
also be used as the label agent. These proteins are normal
constituents of the cell walls of streptococcal bacteria. They
exhibit a strong non-immunogenic reactivity with immunoglobulin
constant regions from a variety of species (see, generally Kronval,
et al. (1973) J. Immunol., 111: 1401-1406, and Akerstrom (1985) J.
Immunol., 135: 2589-2542).
[0119] Typical immunoassays for detecting the target polypeptide(s)
are either competitive or noncompetitive. Noncompetitive
immunoassays are assays in which the amount of captured analyte is
directly measured. In one "sandwich" assay, for example, the
capture agents (antibodies) can be bound directly to a solid
substrate where they are immobilized. These immobilized antibodies
then capture the target polypeptide present in the test sample. The
target polypeptide thus immobilized is then bound by a labeling
agent, such as a second antibody bearing a label.
[0120] In competitive assays, the amount of analyte (MYLIP
polypeptide) present in the sample is measured indirectly by
measuring the amount of an added (exogenous) analyte displaced (or
competed away) from a capture agent (antibody) by the analyte
present in the sample. In one competitive assay, a known amount of,
in this case, labeled polypeptide is added to the sample and the
sample is then contacted with a capture agent. The amount of
labeled polypeptide bound to the antibody is inversely proportional
to the concentration of target polypeptide present in the
sample.
[0121] In one embodiment, the antibody is immobilized on a solid
substrate. The amount of target polypeptide bound to the antibody
may be determined either by measuring the amount of target
polypeptide present in an polypeptide/antibody complex, or
alternatively by measuring the amount of remaining uncomplexed
polypeptide.
[0122] The immunoassay methods of the present invention include an
enzyme immunoassay (EIA) which utilizes, depending on the
particular protocol employed, unlabeled or labeled (e.g.,
enzyme-labeled) derivatives of polyclonal or monoclonal antibodies
or antibody fragments or single-chain antibodies that bind Mylip
polypeptide(s), either alone or in combination. In the case where
the antibody that binds Mylip polypeptide is not labeled, a
different detectable marker, for example, an enzyme-labeled
antibody capable of binding to the monoclonal antibody which binds
the Mylip polypeptide, may be employed. Any of the known
modifications of EIA, for example, enzyme-linked immunoabsorbent
assay (ELISA), may also be employed. As indicated above, also
contemplated by the present invention are immunoblotting
immunoassay techniques such as western blotting employing an
enzymatic detection system.
[0123] The immunoassay methods of the present invention may also be
other known immunoassay methods, for example, fluorescent
immunoassays using antibody conjugates or antigen conjugates of
fluorescent substances such as fluorescein or rhodamine, latex
agglutination with antibody-coated or antigen-coated latex
particles, haemagglutination with antibody-coated or antigen-coated
red blood corpuscles, and immunoassays employing an avidin-biotin
or strepavidin-biotin detection systems, and the like.
[0124] The particular parameters employed in the immunoassays of
the present invention can vary widely depending on various factors
such as the concentration of antigen in the sample, the nature of
the sample, the type of immunoassay employed and the like. Optimal
conditions can be readily established by those of ordinary skill in
the art. In certain embodiments, the amount of antibody that binds
Mylip polypeptides is typically selected to give 50% binding of
detectable marker in the absence of sample. If purified antibody is
used as the antibody source, the amount of antibody used per assay
will generally range from about 1 ng to about 100 ng. Typical assay
conditions include a temperature range of about 4.degree. C. to
about 45.degree. C., preferably about 25.degree. C. to about
37.degree. C., and most preferably about 25.degree. C., a pH value
range of about 5 to 9, preferably about 7, and an ionic strength
varying from that of distilled water to that of about 0.2M sodium
chloride, preferably about that of 0.15M sodium chloride. Times
will vary widely depending upon the nature of the assay, and
generally range from about 0.1 minute to about 24 hours. A wide
variety of buffers, for example PBS, may be employed, and other
reagents such as salt to enhance ionic strength, proteins such as
serum albumins, stabilizers, biocides and non-ionic detergents may
also be included.
[0125] The assays of this invention are scored (as positive or
negative or quantity of target polypeptide) according to standard
methods well known to those of skill in the art. The particular
method of scoring will depend on the assay format and choice of
label. For example, a Western Blot assay can be scored by
visualizing the colored product produced by the enzymatic label. A
clearly visible colored band or spot at the correct molecular
weight is scored as a positive result, while the absence of a
clearly visible spot or band is scored as a negative. The intensity
of the band or spot can provide a quantitative measure of target
polypeptide concentration.
[0126] Antibodies for use in the various immunoassays described
herein can be routinely produced or obtained commercially.
[0127] Idol/LDLR Interaction Assays.
[0128] As shown in Example 2, the residues conserved between LDLR,
VLDLR and apoER2 are important for Idol recognition. It was further
determined that the FERM domain of Idol is important for
interaction with the LDLR. Disruption of Idol FERM domain
interaction with these LDLR residues using a small molecule would
inactivate the Idol-LDLR pathway.
[0129] The Idol-LDLR recognition sequence can be used as the basis
for screens aimed at identifying small molecules that specifically
disrupted Idol-LDLR interaction e.g., by targeting this region of
the LDLR.
[0130] Accordingly, in certain embodiments, screening systems are
contemplated that screen for the ability of test agents to bind the
FERM domain of LDLR and/or to bind/interact with the region of LDLR
that interacts with Idol and/or that inhibit the interaction of
Idol and LDLR.
[0131] In view of the teachings provided herein, methods of
screening for agents that bind the FERM domain of Idol or that bind
to the LDLR region that interacts with Idol are readily available
to those of skill in the art. For example, in certain illustrative
embodiments, Idol FERM domain and/or LDLR domains are immobilized
and probed with test agents. Detection of the test agent (e.g., via
a label attached to the test agent) indicates that the agent binds
to the target moiety and is a good candidate modulator of Idol/LDLR
interaction.
[0132] In another illustrative embodiment, the association of LDLR
and Idol or a FERM domain of Idol in the presence of one or more
test agents is assayed. This can be accomplished using for example
a fluorescence resonance energy transfer system (FRET) comprising a
donor fluorophore on one moiety (e.g., LDLR) and an acceptor
fluorophore on the Idol molecule. The donor and acceptor quench
each other when brought into proximity by the interaction of LDLR
and Idol. When association is reduced or prevented by a test agent
the FRET signal increases indicating that the test agent inhibits
interaction of LDLR and Idol.
[0133] These assays are illustrative and not limiting. Using the
teaching provided herein, numerous binding and/or LDLR/Idol
interaction assays will be available to one of skill in the
art.
[0134] Animal-Based Assays.
[0135] In certain embodiments cells, tissues, and/or animals are
provided that are transfected with an Idol-encoding construct so
they overexpress Idol. In other embodiments, cells, tissues, and/or
animals in which Idol is "knocked out" are provided. It is
completed that one or both of these constructs can be used in
screens for Mylip (Idol) modulators.
[0136] For example, in certain embodiments, test agent(s) (e.g.,
small molecules) are screened for their erect on the Idol pathway
based on the Idol-/- cells, tissues or animals. WT and Idol-/- are
screened for response to candidate small molecules. The effect of
Idol-specific small molecules will be lost in the Idol-/- cells.
These screening methods can be used, for example, in conjunction
with the cell-based reporter screens described herein.
[0137] In certain embodiments, knockout Mylip (Idol) animals are
used in screens for modulators.
[0138] Assay Optimization.
[0139] The assays of this invention have immediate utility in
screening for agents that inhibit Mylip expression and/or activity
in a cell, tissue or organism. The assays of this invention can be
optimized for use in particular contexts, depending, for example,
on the source and/or nature of the biological sample and/or the
particular test agents, and/or the analytic facilities available.
Thus, for example, optimization can involve determining optimal
conditions for binding assays, optimum sample processing conditions
(e.g. preferred PCR conditions), hybridization conditions that
maximize signal to noise, protocols that improve throughput, etc.
In addition, assay formats can be selected and/or optimized
according to the availability of equipment and/or reagents. Thus,
for example, where commercial antibodies or ELISA kits are
available it may be desired to assay protein concentration.
Conversely, where it is desired to screen for modulators that alter
transcription of Mylip gene, nucleic acid based assays are
preferred.
[0140] Routine selection and optimization of assay formats is well
known to those of ordinary skill in the art.
[0141] Pre-Screening for Agents that Bind Mylip.
[0142] In certain embodiments it is desired to pre-screen test
agents for the ability to interact with (e.g. specifically bind to)
a Mylip (or mutant/allele) nucleic acid or polypeptide.
Specifically, binding test agents are more likely to interact with
and thereby modulate Mylip expression and/or activity. Thus, in
some embodiments, the test agent(s) are pre-screened for binding to
Mylip nucleic acids or to Mylip proteins before performing the more
complex assays described above.
[0143] In one embodiment, such pre-screening is accomplished with
simple binding assays. Means of assaying for specific binding or
the binding affinity of a particular ligand for a nucleic acid or
for a protein are well known to those of skill in the art. In
certain illustrative binding assays, the Mylip protein (e.g., Idol
FERM domain or full-length Idol) or nucleic acid encoding such is
immobilized and exposed to a test agent (which can be labeled), or
alternatively, the test agent(s) are immobilized and exposed to an
Mylip protein or to a Mylip nucleic acid (which can be labeled).
The immobilized moiety is then washed to remove any unbound
material and the bound test agent or bound Mylip nucleic acid or
protein is detected (e.g. by detection of a label attached to the
bound molecule). The amount of immobilized label is proportional to
the degree of binding between the Mylip protein or nucleic acid and
the test agent.
[0144] Scoring the Assay(s).
[0145] The assays of this invention are scored according to
standard methods well known to those of skill in the art. The
assays of this invention are typically scored as positive where
there is a difference between the activity seen with the test agent
present or where the test agent has been previously applied, and
the (usually negative) control. In preferred embodiments, the
change is a statistically significant change, e.g. as determined
using any statistical test suited for the data set provided (e.g.
t-test, analysis of variance (ANOVA), semiparametric techniques,
non-parametric techniques (e.g. Wilcoxon Mann-Whitney Test,
Wilcoxon Signed Ranks Test, Sign Test, Kruskal-Wallis Test, etc.).
Preferably the statistically significant change is significant at
least at the 85%, more preferably at least at the 90%, still more
preferably at least at the 95%, and most preferably at least at the
98% or 99% confidence level). In certain embodiments, the change is
at least a 10% change, preferably at least a 20% change, more
preferably at least a 50% change and most preferably at least a 90%
change.
[0146] Agents for Screening: Combinatorial Libraries (e.g., Small
Organic Molecules)
[0147] Virtually any agent can be screened according to the methods
of this invention. Such agents include, but are not limited to
nucleic acids, proteins, sugars, polysaccharides, glycoproteins,
lipids, and small organic molecules. The term small organic
molecule typically refers to molecules of a size comparable to
those organic molecules generally used in pharmaceuticals. The term
excludes biological macromolecules (e.g., proteins, nucleic acids,
etc.). Preferred small organic molecules can range in size up to
about 5000 Da, more preferably up to 2000 Da, and most preferably
up to about 1000 Da.
[0148] Conventionally, new chemical entities with useful properties
are generated by identifying a chemical compound (called a "lead
compound") with some desirable property or activity, creating
variants of the lead compound, and evaluating the property and
activity of those variant compounds. However, the current trend is
to shorten the time scale for all aspects of drug discovery.
Because of the ability to test large numbers quickly and
efficiently, high throughput screening (HTS) methods are replacing
conventional lead compound identification methods.
[0149] In one preferred embodiment, high throughput screening
methods involve providing a library containing a large number of
potential therapeutic compounds (candidate compounds). Such
"combinatorial chemical libraries" are then screened in one or more
assays, as described herein to identify those library members
(particular chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
[0150] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide (e.g., mutein) library is
formed by combining a set of chemical building blocks called amino
acids in every possible way for a given compound length (i.e., the
number of amino acids in a polypeptide compound). Millions of
chemical compounds can be synthesized through such combinatorial
mixing of chemical building blocks. For example, one commentator
has observed that the systematic, combinatorial mixing of 100
interchangeable chemical building blocks results in the theoretical
synthesis of 100 million tetrameric compounds or 10 billion
pentameric compounds (Gallop et al. (1994) 37(9): 1233-1250).
[0151] Preparation of combinatorial chemical libraries is well
known to those of skill in the art. Such combinatorial chemical
libraries include, but are not limited to, peptide libraries (see,
e.g., U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot.
Res., 37: 487-493, Houghton et al. (1991) Nature, 354: 84-88).
Peptide synthesis is by no means the only approach envisioned and
intended for use with the present invention. Other chemistries for
generating chemical diversity libraries can also be used. Such
chemistries include, but are not limited to: peptoids (PCT
Publication No WO 91/19735, 26 Dec. 1991), encoded peptides (PCT
Publication WO 93/20242, 14 Oct. 1993), random bio-oligomers (PCT
Publication WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat.
No. 5,288,514), diversomers such as hydantoins, benzodiazepines and
dipeptides (Hobbs et al., (1993) Proc. Nat. Acad. Sci. USA 90:
6909-6913), vinylogous polypeptides (Hagihara et al. (1992) J.
Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimetics with a
Beta-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.
Chem. Soc. 114: 9217-9218), analogous organic syntheses of small
compound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116:
2661), oligocarbamates (Cho, et al., (1993) Science 261:1303),
and/or peptidyl phosphonates (Campbell et al., (1994) J. Org. Chem.
59: 658). See, generally, Gordon et al., (1994) J. Med. Chem.
37:1385, nucleic acid libraries (see, e.g., Strategene, Corp.),
peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083)
antibody libraries (see, e.g., Vaughn et al. (1996) Nature
Biotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydrate
libraries (see, e.g., Liang et al. (1996) Science, 274: 1520-1522,
and U.S. Pat. No. 5,593,853), and small organic molecule libraries
(see, e.g., benzodiazepines, Baum (1993) C&EN, Jan 18, page 33,
isoprenoids U.S. Pat. No. 5,569,588, thiazolidinones and
metathiazanones U.S. Pat. No. 5,549,974, pyrrolidines U.S. Pat.
Nos. 5,525,735 and 5,519,134, morpholino compounds U.S. Pat. No.
5,506,337, benzodiazepines U.S. Pat. No. 5,288,514, and the
like).
[0152] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.).
[0153] A number of well known robotic systems have also been
developed for solution phase chemistries. These systems include,
but are not limited to, automated workstations like the automated
synthesis apparatus developed by Takeda Chemical Industries, LTD.
(Osaka, Japan) and many robotic systems utilizing robotic arms
(Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,
Hewlett-Packard, Palo Alto, Calif.) which mimic the manual
synthetic operations performed by a chemist and the Venture.TM.
platform, an ultra-high-throughput synthesizer that can run between
576 and 9,600 simultaneous reactions from start to finish (see
Advanced ChemTech, Inc. Louisville, Ky.)). Any of the above devices
are suitable for use with the present invention. The nature and
implementation of modifications to these devices (if any) so that
they can operate as discussed herein will be apparent to persons
skilled in the relevant art. In addition, numerous combinatorial
libraries are themselves commercially available (see, e.g.,
ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St.
Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton,
Pa., Martek Biosciences, Columbia, Md., etc.).
[0154] High Throughput Screening
[0155] Any of the assays described herein are amenable to
high-throughput screening (HTS). Moreover, the cells utilized in
the methods of this invention need not be contacted with a single
test agent at a time. To the contrary, to facilitate
high-throughput screening, a single cell may be contacted by at
least two, preferably by at least 5, more preferably by at least
10, and most preferably by at least 20 test compounds. If the cell
scores positive, it can be subsequently tested with a subset of the
test agents until the agents having the activity are
identified.
[0156] High throughput assays for hybridizaiton assays,
immunoassays, and for various reporter gene products are well known
to those of skill in the art. For example, multi-well fluorimeters
are commercially available (e.g., from Perkin-Elmer).
[0157] In addition, high throughput screening systems are
commercially available (see, e.g., Zymark Corp., Hopkinton, Mass.;
Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc.
Fullerton, Calif.; Precision Systems, Inc., Natick, Mass., etc.).
These systems typically automate entire procedures including all
sample and reagent pipetting, liquid dispensing, timed incubations,
and final readings of the microplate in detector(s) appropriate for
the assay. These configurable systems provide high throughput and
rapid start up as well as a high degree of flexibility and
customization. The manufacturers of such systems provide detailed
protocols the various high throughput. Thus, for example, Zymark
Corp. provides technical bulletins describing screening systems for
detecting the modulation of gene transcription, ligand binding, and
the like.
[0158] Modulator Databases.
[0159] In certain embodiments, the agents that score positively in
the assays described herein (e.g. show an ability to inhibit Mylip
expression and/or activity) can be entered into a database of
putative and/or actual Mylip inhibitors and/or aging inhibitors.
The term database refers to a means for recording and retrieving
information. In preferred embodiments the database also provides
means for sorting and/or searching the stored information. The
database can comprise any convenient media including, but not
limited to, paper systems, card systems, mechanical systems,
electronic systems, optical systems, magnetic systems or
combinations thereof. Preferred databases include electronic (e.g.
computer-based) databases. Computer systems for use in storage and
manipulation of databases are well known to those of skill in the
art and include, but are not limited to "personal computer
systems", mainframe systems, distributed nodes on an inter- or
intra-net, data or databases stored in specialized hardware (e.g.
in microchips), and the like.
II. Inhibition of Mylip.
[0160] It was a surprising discovery that inhibition of Mylip
expression or activity can inhibit LDL receptor degradation and/or
promote LDL uptake in a mammal, and/or mitigate one or more
symptoms of hypercholesterolemia. Any of a variety of methods to
inhibit Mylip can be used.
[0161] RNAi Inhibition of Mylip/Idol.
[0162] Post-transcriptional gene silencing (PTGS) or RNA
interference (RNAi) refers to a mechanism by which double-stranded
(sense strand) RNA (dsRNA) specifically blocks expression of its
homologous gene when injected, or otherwise introduced into cells.
The discovery of this incidence came with the observation that
injection of antisense or sense RNA strands into Caenorhabditis
elegans cells resulted in gene-specific inactivation (Guo and
Kempheus (1995) Cell 81: 611-620). While gene inactivation by the
antisense strand was expected, gene silencing by the sense strand
came as a surprise. Adding to the surprise was the finding that
this gene-specific inactivation actually came from trace amounts of
contaminating dsRNA (Fire et al. (1998) Nature 391: 806-811).
[0163] Since then, this mode of post-transcriptional gene silencing
has been tied to a wide variety of organisms: plants, flies,
trypanosomes, planaria, hydra, zebrafish, and mice (Zamore et al.
(2000). Cell 101: 25-33; Gura (2000) Nature 404: 804-808). RNAi
activity has been associated with functions as disparate as
transposon-silencing, anti-viral defense mechanisms, and gene
regulation (Grant (1999) Cell 96: 303-306).
[0164] By injecting dsRNA into tissues, one can inactivate specific
genes not only in those tissues, but also during various stages of
development. This is in contrast to tissue-specific knockouts or
tissue-specific dominant-negative gene expressions, which do not
allow for gene silencing during various stages of the developmental
process (see, e.g., Gura (2000) Nature 404: 804-808). The
double-stranded RNA is cut by a nuclease activity into 21-23
nucleotide fragments. These fragments, in turn, target the
homologous region of their corresponding mRNA, hybridize, and
result in a double-stranded substrate for a nuclease that degrades
it into fragments of the same size (Hammond et al. (2000) Nature,
404: 293-298; Zamore et al. (2000). Cell 101: 25-33).
[0165] It has been shown that when short (18-30 bp) RNA duplexes
are introduced into mammalian cells in culture, sequence-specific
inhibition of target mRNA can be realized without inducing an
interferon response. Certain of these short dsRNAs, referred to as
small inhibitory RNAs ("siRNAs"), can act catalytically at
sub-molar concentrations to cleave greater than 95% of the target
mRNA in the cell. A description of the mechanisms for siRNA
activity, as well as some of its applications are described in
Provost et al. (2002) EMBO J., 21(21): 5864-5874; Tabara et al.
(2002) Cell 109(7):861-71; Martinez et al. (2002) Cell 110(5): 563;
Hutvagner and Zamore (2002), Science 297: 2056, and the like.
[0166] Using the known nucleotide sequence for the Mylip/Idol gene
and/or mRNA, Mylip/Idol siRNAs can readily be produced. In various
embodiments siRNA that inhibit Mylip/Idol can comprise partially
purified RNA, substantially pure RNA, synthetic RNA, recombinantly
produced RNA, as well as altered RNA that differs from
naturally-occurring RNA by the addition, deletion, substitution
and/or alteration of one or more nucleotides. Such alterations can
include, for example, addition of non-nucleotide material, such as
to the end(s) of the siRNA or to one or more internal nucleotides
of the siRNA, including modifications that make the siRNA resistant
to nuclease digestion.
[0167] In various embodiments one or both strands of the siRNA can
comprise a 3' overhang. As used herein, a "3' overhang" refers to
at least one unpaired nucleotide extending from the 3'-end of an
RNA strand. Thus in one embodiment, the siRNA comprises at least
one 3' overhang of from 1 to about 6 nucleotides (which includes
ribonucleotides or deoxynucleotides) in length, from 1 to about 5
nucleotides in length, from 1 to about 4 nucleotides in length, or
about 2 to about 4 nucleotides in length.
[0168] In an illustrative embodiment in which both strands of the
siRNA molecule comprise a 3' overhang, the length of the overhangs
can be the same or different for each strand. In certain
embodiments the 3' overhang is present on both strands of the
siRNA, and is one, two, or three nucleotides in length. For
example, each strand of the siRNA can comprise 3' overhangs of
dithymidylic acid ("TT") or diuridylic acid ("uu").
[0169] In order to enhance the stability of the siRNA, the 3'
overhangs can be also stabilized against degradation. In one
embodiment, the overhangs are stabilized by including purine
nucleotides, such as adenosine or guanosine nucleotides. In certain
embodiments substitution of pyrimidine nucleotides by modified
analogues, e.g., substitution of uridine nucleotides in the 3'
overhangs with 2'-deoxythymidine, is tolerated and does not affect
the efficiency of RNAi degradation. In particular, it is believed
the absence of a 2' hydroxyl in the 2'-deoxythymidine can
significantly enhance the nuclease resistance of the 3'
overhang
[0170] In certain embodiments, the siRNA comprises the sequence
AA(N19)TT (SEQ ID NO:16), AA(N21)TT (SEQ ID NO:17), NA(N21) (SEQ ID
NO:18), and the like, where N is any nucleotide. In various
embodiments these siRNA comprise approximately 30%-70% GC, and
preferably comprise approximately 50% G/C. The sequence of the
sense siRNA strand corresponds to (N19)TT or N21 (i.e., positions 3
to 23), respectively. In the latter case, the 3' end of the sense
siRNA is converted to TT. The rationale for this sequence
conversion is to generate a symmetric duplex with respect to the
sequence composition of the sense and antisense strand 3'
overhangs. The antisense RNA strand is then synthesized as the
complement to positions 1 to 21 of the sense strand.
[0171] Because position 1 of the 23-nt sense strand in these
embodiments is not recognized in a sequence-specific manner by the
antisense strand, the 3'-most nucleotide residue of the antisense
strand can be chosen deliberately. However, the penultimate
nucleotide of the antisense strand (complementary to position 2 of
the 23-nt sense strand in either embodiment) is generally
complementary to the targeted sequence.
[0172] In another illustrative embodiment, the siRNA comprises the
sequence NAR(N17)YNN (SEQ ID NO:19), where R is a purine (e.g., A
or G) and Y is a pyrimidine (e.g., C or U/T). The respective 21-nt
sense and antisense RNA strands of this embodiment therefore
generally begin with a purine nucleotide. Such siRNA can be
expressed from pol III expression vectors without a change in
targeting site, as expression of RNAs from pol III promoters is
only believed to be efficient when the first transcribed nucleotide
is a purine.
[0173] In various embodiments the siRNA of the invention can be
targeted to any stretch of approximately 10-30, or 15-25, or 19-25
contiguous nucleotides in any of the target mRNA sequences (the
"target sequence"). Techniques for selecting target sequences for
siRNA are given, for example, in Tuschl et al., "The siRNA User
Guide," revised May 6, 2004. The "siRNA User Guide" is available on
the world wide web at a website maintained by Dr. Thomas Tuschl,
and can be found by accessing the website of Rockefeller University
and searching with the keyword "siRNA." In addition, the "siRNA
User Guide" can be located by performing a google search for "siRNA
User Guide" and can also be found at
"www.rockefeller.edu/labheads/tuschl/sirna.html. Techniques for
selecting target sequences for siRNA and miRNA can also be found in
Sioud (2008) siRNA and miRNA Gene Silencing: From Bench to Bedside
(Methods in Molecular Biology), Humana Press.
[0174] In certain embodiments the sense strand of the present siRNA
comprises a nucleotide sequence identical to any contiguous stretch
of about 19 to about 25 nucleotides in the target Mylip/Idol mRNA.
Generally, a target sequence on the target mRNA can be selected
from a given cDNA sequence corresponding to the target mRNA,
preferably beginning 50 to 100 nucleotides downstream (i.e., in the
3' direction) from the start codon. The target sequence can,
however, be located in the 5' or 3' untranslated regions, or in the
region nearby the start.
[0175] The Mylip/Idol silencing siRNAs can be obtained using a
number of techniques known to those of skill in the art. For
example, the siRNA can be chemically synthesized or recombinantly
produced using methods known in the art, such as the Drosophila in
vitro system described in U.S. published application US
2002/0086356.
[0176] In certain embodiments the siRNAs are chemically synthesized
using appropriately protected ribonucleoside phosphoramidites and a
conventional DNA/RNA synthesizer. The siRNAs can be synthesized as
two separate, complementary RNA molecules, or as a single RNA
molecule with two complementary regions. Commercial suppliers of
synthetic RNA molecules or synthesis reagents include Proligo
(Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA),
Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen
Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA) and
Cruachem (Glasgow, UK). Custom siRNA can be obtained from
commercial suppliers (see, e.g., Thermo Fisher Scientific,
Lafayette Colo.; Qiagen, Valencia, Calif.; Applied Biosystems,
Foster City, Calif.; and the like).
[0177] In certain embodiments siRNA can also be expressed from
recombinant circular or linear DNA plasmids using any suitable
promoter. Suitable promoters for expressing siRNA from a plasmid
include, for example, the U6 or H1 RNA pol III promoter sequences
and the cytomegalovirus promoter. Selection of other suitable
promoters is within the skill in the art. The recombinant plasmids
can also comprise inducible or regulatable promoters for expression
of the siRNA in a particular tissue or in a particular
intracellular environment.
[0178] The siRNA expressed from recombinant plasmids can either be
isolated from cultured cell expression systems by standard
techniques, or can be expressed intracellularly at or near the
target area(s) in vivo. The use of recombinant plasmids to deliver
siRNA to cells in vivo is discussed in more detail below.
[0179] siRNA can be expressed from a recombinant plasmid either as
two separate, complementary RNA molecules, or as a single RNA
molecule with two complementary regions. Selection of plasmids
suitable for expressing siRNAs, methods for inserting nucleic acid
sequences for expressing the siRNA into the plasmid, and methods of
delivering the recombinant plasmid to the cells of interest are
within the skill in the art (see, e.g., Tuschl (2002) Nat.
Biotechnol., 20: 446-448; Brummelkamp et al. (2002) Science 296:
550 553; Miyagishi et al. (2002) Nat. Biotechnol. 20: 497-500;
Paddison et al. (2002) Genes Dev. 16: 948-958; Lee et al. (2002)
Nat. Biotechnol. 20: 500-505; Paul et al. (2002) Nat. Biotechnol.
20: 505-508, and the like).
[0180] In one illustrative embodiment, a plasmid comprising nucleic
acid sequences for expressing an siRNA for inhibiting Mylip/Idol
comprises a sense RNA strand coding sequence in operable connection
with a polyT termination sequence under the control of a human U6
RNA promoter, and an antisense RNA strand coding sequence in
operable connection with a polyT termination sequence under the
control of a human U6 RNA promoter. The plasmid is ultimately
intended for use in producing an recombinant adeno-associated viral
vector comprising the same nucleic acid sequences for expressing
the siRNA
[0181] As used herein, "in operable connection with a polyT
termination sequence" means that the nucleic acid sequences
encoding the sense or antisense strands are adjacent to the polyT
termination signal in the 5' direction or sufficiently close so
that during transcription of the sense or antisense sequences from
the plasmid, the polyT termination signals act to terminate
transcription after the desired product is transcribed.
[0182] As used herein, "under the control" of a promoter means that
the nucleic acid sequences encoding the sense or antisense strands
are located 3' of the promoter, so that the promoter can initiate
transcription of the sense or antisense coding sequences.
[0183] In various embodiments the siRNA can be expressed from
recombinant viral vectors intracellularly at or near the target
site(s) in vivo. The recombinant viral vectors comprise sequences
encoding the siRNA of the invention and any suitable promoter for
expressing the siRNA sequences. Suitable promoters include, but are
not limited to, the U6 or H1 RNA pol III promoter sequences and the
cytomegalovirus promoter. Selection of other suitable promoters is
within the skill in the art. The recombinant viral vectors can also
comprise inducible or regulatable promoters for expression of the
siRNA in a particular tissue or in a particular intracellular
environment. The use of recombinant viral vectors to deliver siRNA
of the invention to cells in vivo is discussed in more detail
below.
[0184] The siRNA can be expressed from a recombinant viral vector
either as two separate, complementary RNA molecules, or as a single
RNA molecule with two complementary regions.
[0185] Any viral vector capable of accepting the coding sequences
for the siRNA molecule(s) to be expressed can be used, for example
vectors derived from adenovirus (AV); adeno-associated virus (AAV);
retroviruses (e.g. lentiviruses (LV), Rhabdoviruses, murine
leukemia virus); herpes virus, and the like. The tropism of the
viral vectors can also be modified by pseudotyping the vectors with
envelope proteins or other surface antigens from other viruses. For
example, an AAV vector can be pseudotyped with surface proteins
from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and
the like.
[0186] Selection of recombinant viral vectors suitable for use in
methods for inserting nucleic acid sequences for expressing the
siRNA into the vector, and methods of delivering the viral vector
to the cells of interest are within the skill in the art (see,
e.g., Domburg (1995) Gene Therap. 2: 301-310; Eglitis (1988)
Biotechniques 6: 608-614; Miller (1990) Hum. Gene Therap. 1: 5-14;
Anderson (1998) Nature 392: 25-30, and the like).
[0187] In certain embodiments suitable viral vectors include those
derived from AV and AAV. In one illustrative embodiment, the siRNA
of the invention is expressed as two separate, complementary
single-stranded RNA molecules from a recombinant AAV vector
comprising, for example, either the U6 or H1 RNA promoters, or the
cytomegalovirus (CMV) promoter. A suitable AV vector for expressing
the siRNA, a method for constructing the recombinant AV vector, and
a method for delivering the vector into target cells, are described
in Xia et al. (2002) Nat. Biotech. 20: 1006 1010.
[0188] Suitable AAV vectors for expressing the siRNA, methods for
constructing the recombinant AV vector, and methods for delivering
the vectors into target cells are also described in Samulski et al.
(1987) J. Virol. 61: 3096-3101; Fisher et al. (1996) J. Virol., 70:
520-532; Samulski et al. (1989) J. Virol. 63: 3822-3826; U.S. Pat.
Nos. 5,252,479 and 5,139,941; International Patent Application Nos.
WO 1994/013788; and WO 1993/024641, and the like.
[0189] The ability of an siRNA containing a given target sequence
to cause RNAi-mediated degradation of the target mRNA can be
evaluated using standard techniques for measuring the levels of RNA
or protein in cells. For example, siRNA can be delivered to
cultured cells, and the levels of target mRNA can be measured by
Northern blot or dot blotting techniques, or by quantitative
RT-PCR. Alternatively, the levels of Mylip/Idol in cells can be
measured by ELISA or Western blot.
[0190] RNAi-mediated degradation of target Mylip/Idol mRNA by an
siRNA containing a given target sequence can also be evaluated with
suitable animal models of aging.
[0191] In certain embodiments the siRNA can be delivered as a small
hairpin RNA or short hairpin RNA (shRNA). shRNA is a sequence of
RNA that makes a tight hairpin turn that can be used to silence
gene expression via RNA interference. In typical embodiments, shRNA
uses a vector introduced into cells and utilizes the U6 promoter to
ensure that the shRNA is always expressed. This vector is usually
passed on to daughter cells, allowing the gene silencing to be
inherited. The shRNA hairpin structure is cleaved by the cellular
machinery into siRNA, which is then bound to the RNA-induced
silencing complex (RISC). This complex binds to and cleaves mRNAs
that match the siRNA that is bound to it.
[0192] The shRNA/siRNA described herein target and cause the
RNAi-mediated degradation of EIF4A, or alternative splice forms,
mutants or cognates thereof. Degradation of the target mRNA by the
present siRNA reduces the production of a functional gene product
from the EIF4A gene. Thus, methods are provided for inhibiting
expression of EIF4A in a subject, comprising administering an
effective amount of an EIF4A siRNA to the subject, such that the
target mRNA is degraded.
[0193] It is understood that the siRNA of described herein can
degrade the target mRNA in substoichiometric amounts. Without
wishing to be bound by any theory, it is believed that the siRNA
described herein cause degradation of the target mRNA in a
catalytic manner. Thus, compared to standard anti-angiogenic
therapies, significantly less siRNA needs to be delivered to have a
therapeutic effect.
[0194] One skilled in the art can readily determine an effective
amount of the siRNA to be administered to a given subject, by
taking into account factors such as the size and weight of the
subject; the age, health and sex of the subject; the route of
administration; and whether the administration is regional or
systemic.
[0195] siRNAs suitable to inhibit Mylip are known to those of skill
and a number are commercially available. Thus, for example, Table
1, illustrates data from the SIGMA.RTM. online catalogue describing
a number of Mylip siRNAs and shRNAs.
TABLE-US-00001 TABLE 1 Information about Mylip and siRNA for Mylip
from SIGMA .RTM. catalogue. Properties Official Symbol MYLIP
Species Homo sapiens Entrez Gene ID 29116 Alternate Symbol MYLIP;
MIR Refseq ID(s) NM_013262 Other Designations band 4.1 superfamily
member BZF-1; cellular modulator of immune recognition (c-MIR)
Accession No. (s) AF006003; AF187016; AF212221; AF258586; AJ420601;
AK026739; AK074391; BC002860; BT007055; NM_013262 Protein ID(s)
NP_037394 Homolog(s) Mylip(mouse); Mylip_predicted(Rat) Function(s)
cytoskeleton; ligase; ubiquitin pathway Available Product(s) siRNA;
MISSION .RTM. shRNA panels; shRNA; MISSION .RTM. siRNA panels
Related Products MISSION .RTM. shRNA Type Description
SHGLY-NM_013262 MISSION .RTM. shRNA Bacterial Glycerol Stock
SHVRS-NM_013262 MISSION .RTM. shRNA Lentiviral Transdution
Particles SHDNA-NM_013262 MISSION .RTM. shRNA Plasmid DNA Product #
Description MISSION .RTM. shRNA panels SH2111 MISSION .RTM. shRNA
Human gene family set, bacterial glycerol stock, ubiquitin ligases
(E1, E2, E3) MISSION .RTM. siRNA panels SI00100 MISSION .RTM. siRNA
Human druggable genome library, 6650 targets SI01100 MISSION .RTM.
siRNA Human gene family panels ligase panel, 949 targets siRNA
Approx. siRNA siRNA ID Refseq ID start Ranking SASI_Hs01_00172371
NM_013262 1418 MISSION .RTM. 1 siRNA SASI_Hs01_00172372 NM_013262
491 MISSION .RTM. 2 siRNA SASI_Hs01_00172373 NM_013262 384 MISSION
.RTM. 3 siRNA SASI_Hs01_00172374 NM_013262 371 MISSION .RTM. 4
siRNA SASI_Hs01_00172375 NM_013262 886 MISSION .RTM. 5 siRNA
SASI_Hs01_00172376 NM_013262 1161 MISSION .RTM. 6 siRNA
SASI_Hs01_00172377 NM_013262 878 MISSION .RTM. 7 siRNA
SASI_Hs01_00172378 NM_013262 639 MISSION .RTM. 8 siRNA
SASI_Hs01_00172379 NM_013262 345 MISSION .RTM. 9 siRNA
SASI_Hs01_00172370 NM_013262 389 MISSION .RTM. 10 siRNA
[0196] Double stranded RNA (dsRNA) can be introduced into cells by
any of a wide variety of means. Such methods include, but are not
limited to lipid-mediated transfection (e.g. using reagents such as
lipofectamine), liposome delivery, dendrimer-mediated transfection,
and gene transfer using a viral (e.g., adenoviral vector) or
bacterial vector. Where the vector expresses (transcribes) a
single-stranded RNA, the vector can be designed to transcribe two
complementary RNA strands that will then hybridize to form a
double-stranded RNA.
[0197] Antisense Approaches.
[0198] In various embodiments Mylip expression can be downregulated
or entirely inhibited by the use of antisense molecules. An
"antisense sequence or antisense nucleic acid" is a nucleic acid
that is complementary to the coding Mylip mRNA nucleic acid
sequence or a subsequence thereof. Binding of the antisense
molecule to the Mylip mRNA interferes with normal translation of
the Mylip transcription factor.
[0199] Thus, in accordance with certain embodiments of this
invention, antisense molecules include oligonucleotides and
oligonucleotide analogs that are hybridizable with Mylip messenger
RNA. This relationship is commonly denominated as "antisense." The
oligonucleotides and oligonucleotide analogs are able to inhibit
the function of the RNA, either its translation into protein, its
translocation into the cytoplasm, or any other activity necessary
to its overall biological function. The failure of the messenger
RNA to perform all or part of its function results in a reduction
or complete inhibition of expression of Mylip polypeptides.
[0200] In the context of this invention, the term "oligonucleotide"
refers to a polynucleotide formed from naturally-occurring bases
and/or cyclofuranosyl groups joined by native phosphodiester bonds.
This term effectively refers to naturally-occurring species or
synthetic species formed from naturally-occurring subunits or their
close homologs. The term "oligonucleotide" may also refer to
moieties which function similarly to oligonucleotides, but which
have non naturally-occurring portions. Thus, oligonucleotides may
have altered sugar moieties or inter-sugar linkages. Exemplary
among these are the phosphorothioate and other sulfur containing
species that are known for use in the art. In accordance with some
preferred embodiments, at least one of the phosphodiester bonds of
the oligonucleotide has been substituted with a structure which
functions to enhance the ability of the compositions to penetrate
into the region of cells where the RNA whose activity is to be
modulated is located. It is preferred that such substitutions
comprise phosphorothioate bonds, methyl phosphonate bonds, or short
chain alkyl or cycloalkyl structures. In accordance with other
preferred embodiments, the phosphodiester bonds are substituted
with structures which are, at once, substantially non-ionic and
non-chiral, or with structures which are chiral and
enantiomerically specific. Persons of ordinary skill in the art
will be able to select other linkages for use in the practice of
the invention.
[0201] In one embodiment, the internucleotide phosphodiester
linkage is replaced with a peptide linkage. Such peptide nucleic
acids tend to show improved stability, penetrate the cell more
easily, and show enhances affinity for their target. Methods of
making peptide nucleic acids are known to those of skill in the art
(see, e.g., U.S. Pat. Nos. 6,015,887, 6,015,710, 5,986,053,
5,977,296, 5,902,786, 5,864,010, 5,786,461, 5,773,571, 5,766,855,
5,736,336, 5,719,262, and 5,714,331).
[0202] Oligonucleotides may also include species that include at
least some modified base forms. Thus, purines and pyrimidines other
than those normally found in nature may be so employed. Similarly,
modifications on the furanosyl portions of the nucleotide subunits
may also be effected, as long as the essential tenets of this
invention are adhered to. Examples of such modifications are
2'-O-alkyl- and 2'-halogen-substituted nucleotides. Some specific
examples of modifications at the 2' position of sugar moieties
which are useful in the present invention are OH, SH, SCH.sub.3, F,
OCH.sub.3, OCN, O(CH.sub.2)[n]NH.sub.2 or O(CH.sub.2)[n]CH.sub.3,
where n is from 1 to about 10, and other substituents having
similar properties.
[0203] Such oligonucleotides are best described as being
functionally interchangeable with natural oligonucleotides or
synthesized oligonucleotides along natural lines, but which have
one or more differences from natural structure. All such analogs
are comprehended by this invention so long as they function
effectively to hybridize with messenger RNA of MYLIP to inhibit the
function of that RNA.
[0204] The oligonucleotides in accordance with certain embodiments
of this invention comprise from about 3 to about 50 subunits. It is
more preferred that such oligonucleotides and analogs comprise from
about 8 to about 25 subunits and still more preferred to have from
about 12 to about 20 subunits. As will be appreciated, a subunit is
a base and sugar combination suitably bound to adjacent subunits
through phosphodiester or other bonds. The oligonucleotides used in
accordance with this invention can be conveniently and routinely
made through the well-known technique of solid phase synthesis.
Equipment for such syntheses is sold by several vendors (e.g.
Applied Biosystems). Any other means for such synthesis may also be
employed, however, the actual synthesis of the oligonucleotides is
well within the talents of the routineer. It is also will known to
prepare other oligonucleotide such as phosphorothioates and
alkylated derivatives.
[0205] Ribozymes.
[0206] In another approach, Mylip expression can be inhibited by
the use of ribozymes. As used herein, "ribozymes" include RNA
molecules that contain antisense sequences for specific
recognition, and an RNA-cleaving enzymatic activity. The catalytic
strand cleaves a specific site in a target (Mylip) RNA, preferably
at greater than stoichiometric concentration. Two "types" of
ribozymes are particularly useful in this invention, the hammerhead
ribozyme (Rossi et al. (1991) Pharmac. Ther. 50: 245-254) and the
hairpin ribozyme (Hampel et al. (1990) Nucl. Acids Res. 18:
299-304, and U.S. Pat. No. 5,254,678).
[0207] Because both hammerhead and hairpin ribozymes are catalytic
molecules having antisense and endoribonucleotidase activity,
ribozyme technology has emerged as a potentially powerful extension
of the antisense approach to gene inactivation. The ribozymes of
the invention typically consist of RNA, but such ribozymes may also
be composed of nucleic acid molecules comprising chimeric nucleic
acid sequences (such as DNA/RNA sequences) and/or nucleic acid
analogs (e.g., phosphorothioates).
[0208] Accordingly, within one aspect of the present invention
ribozymes have the ability to inhibit Mylip expression. Such
ribozymes may be in the form of a "hammerhead" (for example, as
described by Forster and Symons (1987) Cell 48: 211-220,; Haseloff
and Gerlach (1988) Nature 328: 596-600; Walbot and Bruening (1988)
Nature 334: 196; Haseloff and Gerlach (1988) Nature 334: 585) or a
"hairpin" (see, e.g. U.S. Pat. No. 5,254,678 and Hampel et al.,
European Patent Publication No. 0 360 257, published Mar. 26,
1990), and have the ability to specifically target, cleave and
Mylip nucleic acids.
[0209] Ribozymes, as well as DNA encoding such ribozymes and other
suitable nucleic acid molecules can be chemically synthesized using
methods well known in the art for the synthesis of nucleic acid
molecules. Alternatively, Promega, Madison, Wis., USA, provides a
series of protocols suitable for the production of RNA molecules
such as ribozymes. The ribozymes also can be prepared from a DNA
molecule or other nucleic acid molecule (which, upon transcription,
yields an RNA molecule) operably linked to an RNA polymerase
promoter, e.g., the promoter for T7 RNA polymerase or SP6 RNA
polymerase. Such a construct may be referred to as a vector.
Accordingly, also provided by this invention are nucleic acid
molecules, e.g., DNA or cDNA, coding for the ribozymes of this
invention. When the vector also contains an RNA polymerase promoter
operably linked to the DNA molecule, the ribozyme can be produced
in vitro upon incubation with the RNA polymerase and appropriate
nucleotides. In a separate embodiment, the DNA may be inserted into
an expression cassette (see, e.g., Cotten and Birnstiel (1989) EMBO
J. 8(12):3861-3866; Hempel et al. (1989) Biochem. 28: 4929-4933,
etc.).
[0210] After synthesis, the ribozyme can be modified by ligation to
a DNA molecule having the ability to stabilize the ribozyme and
make it resistant to RNase. Alternatively, the ribozyme can be
modified to the phosphothio analog for use in liposome delivery
systems. This modification also renders the ribozyme resistant to
endonuclease activity.
[0211] The ribozyme molecule also can be in a host prokaryotic or
eukaryotic cell in culture or in the cells of an organism/patient.
Appropriate prokaryotic and eukaryotic cells can be transfected
with an appropriate transfer vector containing the DNA molecule
encoding a ribozyme of this invention. Alternatively, the ribozyme
molecule, including nucleic acid molecules encoding the ribozyme,
may be introduced into the host cell using traditional methods such
as transformation using calcium phosphate precipitation (Dubensky
et al. (1984) Proc. Natl. Acad. Sci., USA, 81: 7529-7533), direct
microinjection of such nucleic acid molecules into intact target
cells (Acsadi et al. (1991) Nature 352: 815-818), and
electroporation whereby cells suspended in a conducting solution
are subjected to an intense electric field in order to transiently
polarize the membrane, allowing entry of the nucleic acid
molecules. Other procedures include the use of nucleic acid
molecules linked to an inactive adenovirus (Cotton et al. (1990)
Proc. Natl. Acad. Sci., USA, 89:6094), lipofection (Felgner et al.
(1989) Proc. Natl. Acad. Sci. USA 84: 7413-7417), microprojectile
bombardment (Williams et al. (1991) Proc. Natl. Acad. Sci., USA,
88: 2726-2730), polycation compounds such as polylysine, receptor
specific ligands, liposomes entrapping the nucleic acid molecules,
spheroplast fusion whereby E coli containing the nucleic acid
molecules are stripped of their outer cell walls and fused to
animal cells using polyethylene glycol, viral transduction, (Cline
et al., (1985) Pharmac. Ther. 29: 69; and Friedmann et al. (1989)
Science 244: 1275), and DNA ligand (Wu et al (1989) J. Biol. Chem.
264: 16985-16987), as well as psoralen inactivated viruses such as
Sendai or Adenovirus. In one preferred embodiment, the ribozyme is
introduced into the host cell utilizing a lipid, a liposome or a
retroviral vector.
[0212] When the DNA molecule is operatively linked to a promoter
for RNA transcription, the RNA can be produced in the host cell
when the host cell is grown under suitable conditions favoring
transcription of the DNA molecule. The vector can be, but is not
limited to, a plasmid, a virus, a retrotransposon or a cosmid.
Examples of such vectors are disclosed in U.S. Pat. No. 5,166,320.
Other representative vectors include, but are not limited to
adenoviral vectors (e.g., WO 94/26914, WO 93/9191; Kolls et al.
(1994) PNAS 91(1):215-219; Kass-Eisler et al., (1993) Proc. Natl.
Acad. Sci., USA, 90(24): 11498-502, Guzman et al. (1993)
Circulation 88(6): 2838-48, 1993; Guzman et al. (1993) Cir. Res.
73(6):1202-1207, 1993; Zabner et al. (1993) Cell 75(2): 207-216; Li
et al. (1993) Hum Gene Ther. 4(4): 403-409; Caillaud et al. (1993)
Eur. JNeurosci. 5(10): 1287-1291), adeno-associated vector type 1
("AAV-1") or adeno-associated vector type 2 ("AAV-2") (see WO
95/13365; Flotte et al. (1993) Proc. Natl. Acad. Sci., USA,
90(22):10613-10617), retroviral vectors (e.g., EP 0 415 731; WO
90/07936; WO 91/02805; WO 94/03622; WO 93/25698; WO 93/25234; U.S.
Pat. No. 5,219,740; WO 93/11230; WO 93/10218) and herpes viral
vectors (e.g., U.S. Pat. No. 5,288,641). Methods of utilizing such
vectors in gene therapy are well known in the art, see, for
example, Larrick and Burck (1991) Gene Therapy: Application of
Molecular Biology, Elsevier Science Publishing Co., Inc., New York,
N.Y., and Kreigler (1990) Gene Transfer and Expression: A
Laboratory Manual, W.H. Freeman and Company, New York.
[0213] To produce ribozymes in vivo utilizing vectors, the
nucleotide sequences coding for ribozymes are preferably placed
under the control of a strong promoter such as the lac, SV40 late,
SV40 early, or lambda promoters. Ribozymes are then produced
directly from the transfer vector in vivo.
[0214] Inhibiting Mylip/Idol LDLR Interaction.
[0215] As shown in Example 2, the residues conserved between LDLR,
VLDLR and apoER2 are important for Idol recognition. We have
further determined that the FERM domain of Idol is critical for
interaction with the LDLR. Disruption of Idol FERM domain
interaction with these LDLR residues using a small molecule would
inactivate the Idol-LDLR pathway.
[0216] The Idol-LDLR recognition sequence can be used as the basis
for screens aimed at identifying small molecules that specifically
disrupted Idol-LDLR interaction e.g, by targeting this region of
the LDLR.
[0217] Accordingly in certain embodiment, it is contemplated that
Idol/LDLR inhibition can be achieved by using agents (e.g.,
antibodies, small organic molecules, etc.) that bind the FERM
domain of Idol or that bind/interact with the region of LDLR that
interacts with Idol.
[0218] Mylip Inhibitors
[0219] In certain embodiments, it is contemplated that small
organic molecules can be used in inhibit Mylip expression and/or
activity. It is believed inhibitors identified using, for example,
the screening methods described herein can readily be used to
inhibit Mylip.
III. Modes of Administration.
[0220] The mode of administration of the Mylip inhibitor (agent
that inhibits expression and/or activity of Mylip) depends on the
nature of the particular agent. Antisense molecules, catalytic RNAs
(ribozymes), catalytic DNAs, small organic molecules, RNAi, and
other molecules (e.g. lipids, antibodies, etc.) used as MyLip
inhibitors can be formulated as pharmaceuticals (e.g. with suitable
excipient) and delivered using standard pharmaceutical formulation
and delivery methods as described below. Antisense molecules,
catalytic RNAs (ribozymes), catalytic DNAs, and additionally,
knockout constructs, and constructs encoding intrabodies can be
delivered and (if necessary) expressed in target cells (e.g.
hepatic cells) using methods of gene therapy, e.g. as described
below.
[0221] A) Pharmaceutical Formulations.
[0222] The compositions of the invention include bulk drug
compositions useful in the manufacture of non-pharmaceutical
compositions (e.g., impure or non-sterile compositions) and
pharmaceutical compositions (i.e., compositions that are suitable
for administration to a subject or patient) that can be used
directly and/or in the preparation of unit dosage forms. Such
compositions comprise a therapeutically effective amount of one or
more therapeutic agents (e.g. Mylip inhibitors) disclosed herein or
a combination of the agent(s) and a pharmaceutically acceptable
carrier. Preferably, compositions of the invention comprise a
therapeutically effective amount of an inhibitor of expression
and/or activity of Mylip inhibitors and optionally, a
pharmaceutically acceptable carrier.
[0223] The agents that inhibit expression or activity of Mylip used
in the methods of this invention, (e.g. to mitigate one or more
symptoms of hypercholesterolemia) can be prepared and administered
in a wide variety of rectal, oral and parenteral dosage forms for
treating and preventing neurological damage, increased vascular
permeability associated with trauma, and the like. One or more
Mylip inhibitors can be administered by injection, that is,
intravenously, intramuscularly, intracutaneously, subcutaneously,
intraduodenally, or intraperitoneally. Also, the compounds can be
administered by inhalation, for example, intranasally.
Additionally, the compounds can be administered transdermally.
[0224] In certain embodiments, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans, or suitable for administration to an animal
or human. The term "carrier" refers to a diluent, adjuvant (e.g.,
Freund's adjuvant (complete and incomplete)), excipient, or vehicle
with which the therapeutic is administered. Such pharmaceutical
carriers can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water is a preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. The composition, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents. These compositions can take the
form of solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations and the like.
[0225] Generally, the ingredients of the compositions of the
invention are supplied either separately or mixed together in unit
dosage form, for example, as a dry lyophilized powder or water free
concentrate in a hermetically sealed container such as an ampoule
or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed
with an infusion bottle containing sterile pharmaceutical grade
water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0226] The compositions of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include
those formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0227] Pharmaceutical compositions comprising the inhibitors of
Mylip expression and/or activity can be manufactured by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes. Pharmaceutical compositions may be formulated in
conventional manner using one or more physiologically acceptable
carriers, diluents, excipients or auxiliaries that facilitate
processing of the molecules into preparations that can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0228] For topical or transdermal administration, the Mylip
inhibitors can be formulated as solutions, gels, ointments, creams,
lotion, emulsion, suspensions, etc. as are well-known in the art.
Systemic formulations include those designed for administration by
injection, e.g. subcutaneous, intravenous, intramuscular,
intrathecal or intraperitoneal injection, as well as those designed
for transdermal, transmucosal, inhalation, oral or pulmonary
administration.
[0229] For injection, the Mylip inhibitors an be formulated in
aqueous solutions, e.g., in physiologically compatible buffers such
as Hanks's solution, Ringer's solution, or physiological saline
buffer. The solution can contain formulatory agents such as
suspending, stabilizing and/or dispersing agents. Alternatively,
compositions comprising the inhibitors of Mylip expression and/or
activity can be in powder form for constitution with a suitable
vehicle, e.g., sterile pyrogen-free water, before use.
[0230] For transmucosal administration, penetrants appropriate to
the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
[0231] For oral administration, the Mylip inhibitors can be readily
formulated by combining the inhibitors with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
inhibitors of to be formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions and the
like, for oral ingestion by a patient to be treated. For oral solid
formulations such as, for example, powders, capsules and tablets,
suitable excipients include fillers such as sugars, e.g. lactose,
sucrose, mannitol and sorbitol; cellulose preparations such as
maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP);
granulating agents; and binding agents. If desired, disintegrating
agents may be added, such as the cross-linked polyvinylpyrrolidone,
agar, or alginic acid or a salt thereof such as sodium
alginate.
[0232] If desired, solid dosage forms may be sugar-coated or
enteric-coated using standard techniques.
[0233] For oral liquid preparations such as, for example,
suspensions, elixirs and solutions, suitable carriers, excipients
or diluents include water, glycols, oils, alcohols, etc.
Additionally, flavoring agents, preservatives, coloring agents and
the like can be added.
[0234] For buccal administration, the Mylip inhibitors can take the
form of tablets, lozenges, etc. formulated in conventional
manner.
[0235] For administration by inhalation, the Mylip inhibitors for
use according to the present invention are conveniently delivered
in the form of an aerosol spray from pressurized packs or a
nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges of gelatin for use in an inhaler or
insufflator may be formulated containing a powder mix of the Mylip
inhibitors and a suitable powder base such as lactose or
starch.
[0236] The Mylip inhibitors can also be formulated in rectal or
vaginal compositions such as suppositories or retention enemas,
e.g., containing conventional suppository bases
[0237] In various embodiments other pharmaceutical delivery systems
can be employed. Liposomes and emulsions are well known examples of
delivery vehicles that may be used to deliver the Mylip inhibitors.
Certain organic solvents such as dimethylsulfoxide also may be
employed, although usually at the cost of greater toxicity.
Additionally, the Mylip inhibitors can be delivered using a
sustained-release system, such as semipermeable matrices of solid
polymers containing the therapeutic agent. Various
sustained-release materials have been established and are well
known by those skilled in the art. Sustained-release capsules may,
depending on their chemical nature, release the Mylip inhibitors
for a few days, a few weeks, or up to over 100 days. Depending on
the chemical nature and the biological stability of the inhibitors
additional strategies for stabilization can be employed.
[0238] B) "Genetic" Delivery Methods.
[0239] As indicated above, antisense molecules, catalytic RNAs
(ribozymes), catalytic DNAs, RNAi, and additionally, knockout
constructs, and constructs encoding intrabodies can be delivered
and transcribed and/or expressed in target cells (e.g. hepatic
cells) using methods of gene therapy. Thus, in certain preferred
embodiments, the nucleic acids encoding knockout constructs,
intrabodies, antisense molecules, catalytic RNAs or DNAs, etc. are
cloned into gene therapy vectors that are competent to transfect
cells (such as human or other mammalian cells) in vitro and/or in
vivo.
[0240] Many approaches for introducing nucleic acids into cells in
vivo, ex vivo and in vitro are known. These include lipid or
liposome based gene delivery (WO 96/18372; WO 93/24640; Mannino and
Gould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose U.S. Pat.
No. 5,279,833; WO 91/06309; and Felgner et al. (1987) Proc. Natl.
Acad. Sci. USA 84: 7413-7414) and replication-defective retroviral
vectors harboring a therapeutic polynucleotide sequence as part of
the retroviral genome (see, e.g., Miller et al. (1990) Mol. Cell.
Biol. 10:4239 (1990); Kolberg (1992) J. NIH Res. 4: 43, and
Cornetta et al. (1991) Hum. Gene Ther. 2: 215).
[0241] For a review of gene therapy procedures, see, e.g.,
Anderson, Science (1992) 256: 808-813; Nabel and Felgner (1993)
TIBTECH 11: 211-217; Mitani and Caskey (1993) TIBTECH 11: 162-166;
Mulligan (1993) Science, 926-932; Dillon (1993) TIBTECH 11:
167-175; Miller (1992) Nature 357: 455-460; Van Brunt (1988)
Biotechnology 6(10): 1149-1154; Vigne (1995) Restorative Neurology
and Neuroscience 8: 35-36; Kremer and Perricaudet (1995) British
Medical Bulletin 51(1) 31-44; Haddada et al. (1995) in Current
Topics in Microbiology and Immunology, Doerfler and Bohm (eds)
Springer-Verlag, Heidelberg Germany; and Yu et al., (1994) Gene
Therapy, 1:13-26.
[0242] Widely used vector systems include, but are not limited to
adenovirus, adeno associated virus, and various retroviral
expression systems. The use of adenoviral vectors is well known to
those of skill and is described in detail, e.g., in WO 96/25507.
Particularly preferred adenoviral vectors are described by Wills et
al. (1994) Hum. Gene Therap. 5: 1079-1088.
[0243] Adeno-associated virus (AAV)-based vectors used to transduce
cells with target nucleic acids, e.g., in the in vitro production
of nucleic acids and peptides, and in in vivo and ex vivo gene
therapy procedures are describe, for example, by West et al. (1987)
Virology 160:38-47; Carter et al. (1989) U.S. Pat. No. 4,797,368;
Carter et al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy
5:793-801; Muzyczka (1994) J. Clin. Invst. 94:1351 for an overview
of AAV vectors. Lebkowski, U.S. Pat. No. 5,173,414; Tratschin et
al. (1985) Mol. Cell. Biol. 5(11):3251-3260; Tratschin, et al.
(1984) Mol. Cell. Biol., 4: 2072-2081; Hermonat and Muzyczka (1984)
Proc. Natl. Acad. Sci. USA, 81: 6466-6470; McLaughlin et al. (1988)
and Samulski et al. (1989) J. Virol., 63:03822-3828. Cell lines
that can be transformed by rAAV include those described in
Lebkowski et al. (1988) Mol. Cell. Biol., 8:3988-3996.
[0244] Widely used retroviral vectors include those based upon
murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),
Simian Immunodeficiency virus (SIV), human immunodeficiency virus
(HIV), alphavirus, and combinations thereof (see, e.g., Buchscher
et al. (1992) J. Virol. 66(5) 2731-2739; Johann et al. (1992) J.
Virol. 66 (5):1635-1640 (1992); Sommerfelt et al., (1990) Virol.
176:58-59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et
al., J. Virol. 65:2220-2224 (1991); Wong-Staal et al.,
PCT/US94/05700, and Rosenburg and Fauci (1993) in Fundamental
Immunology, Third Edition Paul (ed) Raven Press, Ltd., New York and
the references therein, and Yu et al. (1994) Gene Therapy, supra;
U.S. Pat. No. 6,008,535, and the like). Other suitable viral
vectors include, but are not limited to herpes virus, lentivirus,
and vaccinia virus.
[0245] Alone, or in combination with viral vectors, a number of
non-viral vectors are also useful for transfecting cells to express
constructs that block or inhibit Mylip expression. Suitable
non-viral vectors include, but are not limited to, plasmids,
cosmids, phagemids, liposomes, water-oil emulsions, polethylene
imines, biolistic pellets/beads, and dendrimers.
[0246] Liposomes were first described in 1965 as a model of
cellular membranes and quickly were applied to the delivery of
substances to cells. Liposomes entrap DNA by one of two mechanisms
which has resulted in their classification as either cationic
liposomes or pH-sensitive liposomes. Cationic liposomes are
positively charged liposomes which interact with the negatively
charged DNA molecules to form a stable complex. Cationic liposomes
typically consist of a positively charged lipid and a co-lipid.
Commonly used co-lipids include dioleoyl phosphatidylethanolamine
(DOPE) or dioleoyl phosphatidylcholine (DOPC). Co-lipids, also
called helper lipids, are in most cases required for stabilization
of liposome complex. A variety of positively charged lipid
formulations are commercially available and many other are under
development. Two of the most frequently cited cationic lipids are
lipofectamine and lipofectin. Lipofectin is a commercially
available cationic lipid first reported by Phil Felgner in 1987 to
deliver genes to cells in culture. Lipofectin is a mixture of
N-[1-(2,3-dioleyloyx)propyl]-N--N--N-trimethyl ammonia chloride
(DOTMA) and DOPE.
[0247] DNA and lipofectin or lipofectamine interact spontaneously
to form complexes that have a 100% loading efficiency. In other
words, essentially all of the DNA is complexed with the lipid,
provided enough lipid is available. It is assumed that the negative
charge of the DNA molecule interacts with the positively charged
groups of the DOTMA. The lipid:DNA ratio and overall lipid
concentrations used in forming these complexes are extremely
important for efficient gene transfer and vary with application.
Lipofectin has been used to deliver linear DNA, plasmid DNA, and
RNA to a variety of cells in culture. Shortly after its
introduction, it was shown that lipofectin could be used to deliver
genes in vivo. Following intravenous administration of
lipofectin-DNA complexes, both the lung and liver showed marked
affinity for uptake of these complexes and transgene expression.
Injection of these complexes into other tissues has had varying
results and, for the most part, are much less efficient than
lipofectin-mediated gene transfer into either the lung or the
liver.
[0248] PH-sensitive, or negatively-charged liposomes, entrap DNA
rather than complex with it. Since both the DNA and the lipid are
similarly charged, repulsion rather than complex formation occurs.
Yet, some DNA does manage to get entrapped within the aqueous
interior of these liposomes. In some cases, these liposomes are
destabilized by low pH and hence the term pH-- sensitive. To date,
cationic liposomes have been much more efficient at gene delivery
both in vivo and in vitro than pH-sensitive liposomes. pH-sensitive
liposomes have the potential to be much more efficient at in vivo
DNA delivery than their cationic counterparts and should be able to
do so with reduced toxicity and interference from serum
protein.
[0249] In another approach dendrimers complexed to the DNA have
been used to transfect cells. Such dendrimers include, but are not
limited to, "starburst" dendrimers and various dendrimer
polycations.
[0250] Dendrimer polycations are three dimensional, highly ordered
oligomeric and/or polymeric compounds typically formed on a core
molecule or designated initiator by reiterative reaction sequences
adding the oligomers and/or polymers and providing an outer surface
that is positively changed. These dendrimers may be prepared as
disclosed in PCT/US83/02052, and U.S. Pat. Nos. 4,507,466,
4,558,120, 4,568,737, 4,587,329, 4,631,337, 4,694,064, 4,713,975,
4,737,550, 4,871,779, 4,857,599.
[0251] Typically, the dendrimer polycations comprise a core
molecule upon which polymers are added. The polymers may be
oligomers or polymers which comprise terminal groups capable of
acquiring a positive charge. Suitable core molecules comprise at
least two reactive residues which can be utilized for the binding
of the core molecule to the oligomers and/or polymers. Examples of
the reactive residues are hydroxyl, ester, amino, imino, imido,
halide, carboxyl, carboxyhalide maleimide, dithiopyridyl, and
sulfhydryl, among others. Preferred core molecules are ammonia,
tris-(2-aminoethyl)amine, lysine, ornithine, pentaerythritol and
ethylenediamine, among others. Combinations of these residues are
also suitable as are other reactive residues.
[0252] Oligomers and polymers suitable for the preparation of the
dendrimer polycations of the invention are
pharmaceutically-acceptable oligomers and/or polymers that are well
accepted in the body. Examples of these are polyamidoamines derived
from the reaction of an alkyl ester of an
.alpha.,.beta.-ethylenically unsaturated carboxylic acid or an
.alpha.,.beta.-ethylenically unsaturated amide and an alkylene
polyamine or a polyalkylene polyamine, among others. Preferred are
methyl acrylate and ethylenediamine. The polymer is preferably
covalently bound to the core molecule.
[0253] The terminal groups that may be attached to the oligomers
and/or polymers should be capable of acquiring a positive charge.
Examples of these are azoles and primary, secondary, tertiary and
quaternary aliphatic and aromatic amines and azoles, which may be
substituted with S or O, guanidinium, and combinations thereof. The
terminal cationic groups are preferably attached in a covalent
manner to the oligomers and/or polymers. Preferred terminal
cationic groups are amines and guanidinium. However, others may
also be utilized. The terminal cationic groups may be present in a
proportion of about 10 to 100% of all terminal groups of the
oligomer and/or polymer, and more preferably about 50 to 100%.
[0254] The dendrimer polycation may also comprise 0 to about 90%
terminal reactive residues other than the cationic groups. Suitable
terminal reactive residues other than the terminal cationic groups
are hydroxyl, cyano, carboxyl, sulfhydryl, amide and thioether,
among others, and combinations thereof. However others may also be
utilized.
[0255] The dendrimer polycation is generally and preferably
non-covalently associated with the polynucleotide. This permits an
easy disassociation or disassembling of the composition once it is
delivered into the cell. Typical dendrimer polycation suitable for
use herein have a molecular weight ranging from about 2,000 to
1,000,000 Da, and more preferably about 5,000 to 500,000 Da.
However, other molecule weights are also suitable. Preferred
dendrimer polycations have a hydrodynamic radius of about 11 to 60
.ANG.., and more preferably about 15 to 55 .ANG.. Other sizes,
however, are also suitable. Methods for the preparation and use of
dendrimers in gene therapy are well known to those of skill in the
art and describe in detail, for example, in U.S. Pat. No.
5,661,025.
[0256] Where appropriate, two or more types of vectors can be used
together. For example, a plasmid vector may be used in conjunction
with liposomes. In the case of non-viral vectors, nucleic acid may
be incorporated into the non-viral vectors by any suitable means
known in the art. For plasmids, this typically involves ligating
the construct into a suitable restriction site. For vectors such as
liposomes, water-oil emulsions, polyethylene amines and dendrimers,
the vector and construct may be associated by mixing under suitable
conditions known in the art.
[0257] C) Effective Dosages.
[0258] The inhibitors of Mylip expression and/or activity will
generally be used in an amount effective to achieve the intended
purpose (e.g. to provided for inhibiting LDL receptor degradation
and/or promoting LDL uptake in a mammal, and/or one mitigating or
more symptoms of hypercholesterolemia in a mammal). In certain
embodiments, the Mylip inhibitors utilized in the methods of this
invention are administered at a dose that is effective to partially
or fully for inhibit LDL receptor degradation and/or promote LDL
uptake in a mammal, and/or one mitigate one or more symptoms of
hypercholesterolemia in a mammal (e.g., a statistically significant
decrease at the 90%, more preferably at the 95%, and most
preferably at the 98% or 99% confidence level). In certain
embodiments the compounds can also be used prophalactically at the
same dose levels.
[0259] Typically, the inhibitors of Mylip expression and/or
activity, or pharmaceutical compositions thereof, are administered
or applied in a therapeutically effective amount. A therapeutically
effective amount is an amount effective that is effective to
partially or fully for inhibit LDL receptor degradation and/or
promote LDL uptake in a mammal, and/or one mitigate one or more
symptoms of hypercholesterolemia in a mammal. Determination of a
therapeutically effective amount is well within the capabilities of
those skilled in the art, especially in light of the detailed
disclosure provided herein.
[0260] For systemic administration, a therapeutically effective
dose can be estimated initially from in vitro assays. For example,
a dose can be formulated in animal models to achieve a circulating
concentration range that includes the IC.sub.50 as determined in
cell culture. Such information can be used to more accurately
determine useful doses in humans.
[0261] Initial dosages can also be estimated from in vivo data,
e.g., animal models, using techniques that are well known in the
art. One skilled in the art could readily optimize administration
to humans based on animal data.
[0262] Dosage amount and interval may be adjusted individually to
provide plasma levels of the inhibitors which are sufficient to
maintain therapeutic effect.
[0263] In certain embodiments, an initial dosage of about 1 .mu.g
preferably from about 1 mg to about 1000 mg per kilogram daily will
be effective. A daily dose range of about 5 to about 75 mg is
preferred. The dosages, however, may be varied depending upon the
requirements of the patient, the severity of the condition being
treated, and the compound being employed. Determination of the
proper dosage for a particular situation is within the skill of the
art. Generally, treatment is initiated with smaller dosages which
are less than the optimum dose of the compound. Thereafter, the
dosage is increased by small increments until the optimum effect
under the circumstance is reached. F or convenience, the total
daily dosage may be divided and administered in portions during the
day if desired. Typical dosages will be from about 0.1 to about 500
mg/kg, and ideally about 25 to about 250 mg/kg.
[0264] In cases of local administration or selective uptake, the
effective local concentration of the inhibitors may not be related
to plasma concentration. One skilled in the art will be able to
optimize therapeutically effective local dosages without undue
experimentation. The amount of inhibitor administered will, of
course, be dependent on the subject being treated, on the subject's
weight, the severity of the affliction, the manner of
administration and the judgment of the prescribing physician.
[0265] The therapy may be repeated intermittently. The therapy may
be provided alone or in combination with other drugs and/or
procedures.
[0266] D) Toxicity.
[0267] Preferably, a therapeutically effective dose of the
inhibitors of Mylip expression and/or activity described herein
will provide therapeutic benefit without causing substantial
toxicity.
[0268] Toxicity of the inhibitors described herein can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., by determining the LD.sub.50 (the
dose lethal to 50% of the population) or the LD.sub.100 (the dose
lethal to 100% of the population). The dose ratio between toxic and
therapeutic effect is the therapeutic index. Inhibitors which
exhibit high therapeutic indices are preferred. The data obtained
from these cell culture assays and animal studies can be used in
formulating a dosage range that is not toxic for use in human. The
dosage of the inhibitors described herein lies preferably within a
range of circulating concentrations that include the effective dose
with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. (See, e.g., Fingl et al., 1975,
In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1).
IV. Mvlip Knock-Out and Knock-In Cells, Tissues, and/or
Animals.
[0269] In certain embodiments, cells, tissues, and/or animals
transfected with a construct that expresses Idol are provided. In
certain embodiments, cells, tissues and/or animals transfected with
a construct that disrupts the Idol gene and thereby reduces Idol
expression are provided.
[0270] Idol Knock-Ins.
[0271] In certain embodiments, cells, tissues, and/or animals
comprising a construct that encodes and expresses Idol are
contemplated. In various embodiments, these cells, tissues and/or
animals, express Idol at a level higher than in the untransfected
cell, tissue, and/or animal. Typically this involves creating a DNA
sequence that encodes Idol, placing the DNA in an expression
cassette under the control of a particular promoter, and
transfecting a cell (in vitro or in vivo) with the expression
cassette whereby the cell expresses the encoded Idol. In various
embodiments, such expression can be constitutive, inducible, or
tissue-specific using constitutive, inducible, or tissue-specific
promoters.
[0272] Methods of expressing heterologous proteins in cells are
well known to those of skill in the art (see, e.g., Sambrook et
al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring
Harbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., Current
Protocols in Molecular Biology (Green Publishing and
Wiley-Interscience: New York, 1987) and periodic updates).
[0273] Idol Knockouts.
[0274] In certain embodiments, animals comprising a disruption of
one or both alleles of an Idol gene (Idol "knockouts") are
provided. The knockout animals of this invention are useful as
systems in which to screen for various agents (e.g. drugs) that
modulate Idol expression and/or activity and thereby modulatge LDL
receptor degredaton and/or LDL uptake.
[0275] The Idol knockouts utilize nucleic acid sequences
(transgenes) that are capable of inactivating endogenous Idol
genes. Such transgenes preferably contain a nucleic acid sequence
(e.g., a DNA sequence) that is identical to some portion of the
endogenous Idol gene that is to be disrupted. Preferred transgenes
of this invention also contain an insertion, deletion, or
substitution of one or more nucleotides; a frameshift mutation;
and/or a stop codon as compared with undisrupted alleles of the
same Idol gene naturally-occurring in the species.
[0276] Homologous recombination of the transgene with a Idol allele
disrupts the expression of that allele. Such a disruption can be by
a number of mechanisms including, but not limited to, interference
in initiation of transcription and/or translation, by premature
termination of transcription and/or translation, and/or by
production of a non-functional Idol protein.
[0277] In one illustrative embodiment, such transgenes are derived
by deleting nucleotides from the nucleic acid sequence encoding the
functional Idol gene. Although the resultant mutated nucleic acid
sequence is incapable of being transcribed and/or translated into a
functional Idol gene product, such transgenes will have sufficient
sequence homology with an endogenous Idol allele of a selected
non-human animal such that the transgene is capable of homologous
recombination with the endogenous Idol allele.
[0278] In one embodiment, transgenes are produced by ligation of an
expression cassette encoding a selectable marker into the nucleic
acid sequence encoding the Idol gene products and/or into the
nucleic acid sequence regulating transcription of the Idol gene
product. The cassette is typically inserted in a location such that
it replaces or disrupts regions of the encoded protein required for
protein functionality. The cassette is also typically inserted in a
location such that splicing out of the cassette introduces a
frameshift mutation resulting in non-functional reversions. In one
embodiment, the expression cassette comprises one or more
selectable marker(s), such as, e.g., .beta.-galactosidase and/or
neomycin phosphotransferase II.
[0279] Such transgenes are preferably designed for replacement of
one or more exons of the endogenous Idol gene. Although insertional
transgenes may also be used, replacement transgenes are preferred
because they significantly reduce the likelihood of secondary
recombination and reversion to the wild-type Idol gene.
[0280] The Idol knockouts of this invention are useful themselves
as models systems for a number of pathologies or can be crossed
with animals exhibiting particular phenotypic traits to produce
useful animal models.
[0281] In certain embodiments, homologous recombination is used to
control the site of integration of a specific DNA sequence
(transgene) into the naturally present Idol sequence of an animal
cell and thereby disrupt that gene and prevent normal its normal
expression (see, e.g., Watson (1977) In: Molecular Biology of the
Gene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif.).
[0282] Typically, this is accomplished using, for example, a
positive/negative selection (PNS) (Thomas and Cappechi (1987) Cell
51: 503-512). This method involves the use of two selectable
markers: one a positive selection marker such as the bacterial gene
for neomycin resistance (neo); the other a negative selection
marker such as the herpes virus thymidine kinase (HSV-tk) gene. Neo
confers resistance to the drug G-418, while HSV-tk renders cells
sensitive to the nucleoside analog gangcyclovir (GANC) or
1-(2-deoxy-2-fluoro-b-d-arabinofuranosyl)-5-iodouraci2l (FIAU). The
DNA encoding the positive selection marker in the transgene (e.g.,
neo) is generally linked to an expression regulation sequence that
allows for its independent transcription in the target cells (e.g.,
embryonic stem (ES) cells). It is flanked by first and second
sequence portions of at least a part of the Idol gene.
[0283] These first and second sequence portions target the
transgene to a specific allele. A second independent expression
unit capable of producing the expression product for a negative
selection marker, e.g., for HSV-tk is positioned adjacent to or in
close proximity to the distal end of the first or second portions
of the first DNA sequence. Upon transfection, some of the ES cells
incorporate the transgene by random integration, others by
homologous recombination between the endogenous allele and
sequences in the transgene. As a result, one copy of the targeted
allele is disrupted by homologous recombination with the-transgene
with simultaneous loss of the sequence encoding herpes HSV-tk gene.
Random integrants, which occur via the ends of the transgene,
contain herpes HSV-tk and remain sensitive to GANC or FIAU.
Therefore, selection, either sequentially or simultaneously with
G418 and GANC enriches for transfected ES cells containing the
transgene integrated into the genome by homologous
recombination.
[0284] Other strategies that select for homologous recombination
events but do not use PNS may also be used.
[0285] It is possible that in some circumstances it will not be
desirable to have an expressed antibiotic resistance gene
incorporated into the knockout animal. Therefore, in certain
embodiments, one or more genetic elements are included in the
knockout construct that permit the antibiotic resistance gene to be
excised once the construct has undergone homologous recombination
with the Idol gene.
[0286] The methods described herein and illustrated in Example 2
are capable of mutating both alleles of the cell's Idol gene;
however, since the frequency of such dual mutational events is the
square of the frequency of a single mutational event, cells having
mutations in both of their Idol alleles will be only a very small
proportion of the total population of mutated cells. It is possible
to readily identify (for example through the use of Southern
hybridization or other methods) whether the mutational events are
single-allele or dual-allele events. Animals having a mutational
event in a single allele may be cross-bred to produce homozygous
animals (having the disruption in both alleles) if the disruption
becomes incorporated in the germ line.
[0287] In one embodiment, the nucleic acid molecule(s) that are to
be introduced into the recipient cell contain a region of homology
with a region of the Idol gene. In a preferred embodiment, the
nucleic acid molecule will contain two regions having homology with
the cell's Idol gene. These "regions of homology" will preferably
flank the precise sequence whose incorporation into the Idol gene
is desired.
[0288] The nucleic acid molecule(s) may be single stranded, but are
preferably double stranded. The molecule(s) may be introduced to
the cell as DNA molecules, as one or more RNA molecules which may
be converted to DNA by reverse transcriptase or by other means.
[0289] To produce the knockout animal, cells are transformed with
the construct (e.g., transgene) described above. As used herein,
the term "transformed" is defined as introduction of exogenous DNA
into the target cell by any means known to the skilled artisan.
These methods of introduction can include, without limitation,
transfection, microinjection, infection (with, for example,
retroviral-based vectors), electroporation and microballistics. The
term "transformed," unless otherwise indicated, is not intended
herein to indicate alterations in cell behavior and growth patterns
accompanying immortalization, density-independent growth, malignant
transformation or similar acquired states in culture.
[0290] To create animals having a particular gene inactivated in
all cells, it is preferable to introduce a knockout construct into
the germ cells (sperm or eggs, i.e., the "germ line") of the
desired species. Genes or other DNA sequences can be introduced
into the pronuclei of fertilized eggs by microinjection or other
methods. Following pronuclear fusion, the developing embryo may
carry the introduced gene in all its somatic and germ cells since
the zygote is the mitotic progenitor of all cells in the embryo.
Since targeted insertion of a knockout construct is a relatively
rare event, it is desirable to generate and screen a large number
of animals when employing such an approach. Because of this, it can
be advantageous to work with the large cell populations and
selection criteria that are characteristic of cultured cell
systems. However, for production of knockout animals from an
initial population of cultured cells, it is preferred that a
cultured cell containing the desired knockout construct be capable
of generating a whole animal. This is generally accomplished by
placing the cell into a developing embryo environment of some
sort.
[0291] Cells capable of giving rise to at least several
differentiated cell types are hereinafter termed "pluripotent"
cells. Pluripotent cells capable of giving rise to all cell types
of an embryo, including germ cells, are hereinafter termed
"totipotent" cells. Totipotent murine cell lines (embryonic stem,
or "ES" cells) have been isolated by culture of cells derived from
very young embryos (blastocysts). Such cells are capable, upon
incorporation into an embryo, of differentiating into all cell
types, including germ cells, and can be employed to generate
animals lacking a functional Idol gene. That is, cultured ES cells
can be transformed with a knockout construct, as described herein,
and cells selected in which the Idol gene is inactivated through
insertion of the construct within the Idol gene.
[0292] The transgenic non-human animals of the invention are
produced by introducing transgenes into the germline of the
non-human animal. Embryonic target cells at various developmental
stages can be used to introduce transgenes. Different methods are
used depending on the stage of development of the embryonic target
cell.
[0293] Microinjection is one illustrative method for transformation
of a zygote. In the mouse, the male pronucleus reaches the size of
approximately 20 micrometers in diameter which allows reproducible
injection of 1-2 .mu.l of DNA solution. The use of zygotes as a
target for gene transfer has a major advantage in that in most
cases the injected DNA will be incorporated into the host gene
before the first cleavage (Brinster et al. (1985) Proc. Natl. Acad.
Sci. USA 82, 4438-4442). As a consequence, all cells of the
transgenic non-human animal will carry the incorporated transgene.
This will, in general, also be reflected in the efficient
transmission of the transgene to offspring of the founder since 50%
of the germ cells will harbor the transgene.
[0294] The gene sequence being introduced need not be incorporated
into any kind of self-replicating plasmid or virus (Jaenisch,
(1988) Science, 240: 1468-1474). Indeed, the presence of vector DNA
has been found, in many cases, to be undesirable (Hammer et al.
(1987) Science 235: 53; Chada et al. (1986) Nature 319: 685;
Kollias et al., (1986) Cell 46: 89; Shani, (1986) Molec, Cell,
Biol. 6: 2624 (1986); Chada, et al. (1985) Nature, 314: 377; Townes
et al. (1985) EMBO J. 4: 1715).
[0295] Once the DNA molecule has been injected into the fertilized
egg cell, the cell is implanted into the uterus of a recipient
female, and allowed to develop into an animal. Since all of the
animal's cells are derived from the implanted fertilized egg, all
of the cells of the resulting animal (including the germ line
cells) contain the introduced gene sequence. If, as occurs in about
30% of events, the first cellular division occurs before the
introduced gene sequence has integrated into the cell's genome, the
resulting animal will be a chimeric animal.
[0296] By breeding and inbreeding such animals, it is possible to
routinely produce heterozygous and homozygous transgenic animals.
Despite any unpredictability in the formation of such transgenic
animals, the animals have generally been found to be stable, and to
be capable of producing offspring that retain and express the
introduced gene sequence.
[0297] The success rate for producing transgenic animals is
greatest in mice. Approximately 25% of fertilized mouse eggs into
which DNA has been injected, and which have been implanted in a
female, will become transgenic mice. A number of other transgenic
animals have also been produced. These include rabbits, sheep,
cattle, and pigs (Jaenisch (1988) Science 240: 1468-1474; Hammer et
al., (1986) J. Animal. Sci, 63: 269; Hammer et al. (1985) Nature
315: 680; Wagner et al., (1984) Theriogenology 21: 29).
[0298] Retroviral infection can also be used to introduce a
transgene into a non-human animal. The developing non-human embryo
can be cultured in vitro to the blastocyst stage. During this time,
the blastomeres can be targets for retroviral infection (Jaenich
(1976) Proc. Natl. Acad. Sci. USA 73: 1260-1264). Efficient
infection of the blastomeres is obtained by enzymatic treatment to
remove the zona pellucida (Hogan, et al. (1986) In Manipulating the
Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.). The viral vector system used to introduce the
transgene is typically a replication-defective retrovirus carrying
the transgene (Jahner, et al. (1985) Proc. Natl. Acad. Sci. USA 82,
6927-6931; Van der Putten, et al. (1985) Proc. Natl. Acad. Sci.,
USA, 82, 6148-6152). Transfection is easily and efficiently
obtained by culturing the blastomeres on a monolayer of
virus-producing cells (Van der Putten, supra; Stewart et al. (1987)
EMBO J., 6: 383-388). Alternatively, infection can be performed at
a later stage. Virus or virus-producing cells can be injected into
the blastocoele (Jahner et al. (1982) Nature, 298: 623-628). Most
of the founders will be mosaic for the transgene since
incorporation occurs only in a subset of the cells, which formed
the transgenic non-human animal. Further, the founder may contain
various retroviral insertions of the transgene at different
positions in the genome which generally will segregate in the
offspring. In addition, it is also possible to introduce transgenes
into the germ line, albeit with low efficiency, by intrauterine
retroviral infection of the midgestation embryo (Jahner et al.
(1982) supra).
[0299] A third and preferred target cell for transgene introduction
is the embryonic stem cell (ES). ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(Evans, et al. (1981) Nature, 292: 154-156; Bradley, et al. (1984)
Nature, 309: 255-258; Gossler, et al. (1986) Proc. Natl. Acad.
Sci., USA, 83: 9065-9069; and Robertson, et al. (1986) Nature, 322:
445-448). Transgenes can be efficiently introduced into the ES
cells a number of means well known to those of skill in the art.
Such transformed ES cells can thereafter be combined with
blastocysts from a non-human animal. The ES cells thereafter
colonize the embryo and contribute to the germ line of the
resulting chimeric animal (for a review see Jaenisch (1988)
Science, 240: 1468-1474).
[0300] The DNA molecule containing the desired gene sequence may be
introduced into the pluripotent cell by any method which will
permit the introduced molecule to undergo recombination at its
regions of homology. Transgenes can be efficiently introduced into
the ES cells by DNA transfection or by retrovirus-mediated
transduction.
[0301] In a particular embodiment, the DNA is introduced by
electroporation (Toneguzzo et al., (1988) Nucleic Acids Res., 16:
5515-5532; Quillet et al. (1988) J. Immunol., 141: 17-20; Machy et
al. (1988) Proc. Natl. Acad. Sci., USA, 85: 8027-8031). After
permitting the introduction of the DNA molecule(s), the cells are
cultured under conventional conditions, as are known in the
art.
[0302] In order to facilitate the recovery of those cells that have
received the DNA molecule containing the desired gene sequence, it
is preferable to introduce the DNA containing the desired gene
sequence in combination with a second gene sequence that would
contain a detectable marker gene sequence. Where it is only desired
to introduce a disruption into a gene, the DNA sequence containing
the detectable marker sequence may itself comprise the disruption.
For the purposes of the present invention, any gene sequence whose
presence in a cell permits one to recognize and clonally isolate
the cell may be employed as a detectable (selectable) marker gene
sequence.
[0303] In one embodiment, the presence of the detectable
(selectable) marker sequence in a recipient cell is recognized by
hybridization, by detection of radiolabelled nucleotides, or by
other assays of detection which do not require the expression of
the detectable marker sequence. In one embodiment, such sequences
are detected using polymerase chain reaction (PCR) or other DNA
amplification techniques to specifically amplify the DNA marker
sequence (Mullis et al., (1986) Cold Spring Harbor Symp. Quant.
Biol. 51: 263-273; Erlich et al. EP 50,424; EP 84,796, EP 258,017
and EP 237,362; Mullis EP 201,184; Mullis et al., U.S. Pat. No.
4,683,202; Erlich U.S. Pat. No. 4,582,788; and Saiki et al. U.S.
Pat. No. 4,683,194).
[0304] Most preferably, however, the detectable marker gene
sequence will be expressed in the recipient cell and will result in
a selectable phenotype. Selectable markers are well known to those
of skill in the art. Some examples include the hprt gene
(Littlefield (1964) Science 145:709-710), the thymidine kinase gene
of herpes simplex virus (Giphart-Gassler et al. (1989) Mutat, Res.,
214: 223-232), the nDtII gene (Thomas et al. (1987) Cell, 51:
503-512; Mansour et al. (1988) Nature 336: 348-352), or other genes
which confer resistance to amino acid or nucleoside analogues, or
antibiotics, etc.
[0305] Thus, for example, cells that express an active HPRT enzyme
are unable to grow in the presence of certain nucleoside analogues
(such as 6-thioguanine, 8-azapurine, etc.), but are able to grow in
media supplemented with HAT (hypoxanthine, aminopterin, and
thymidine). Conversely, cells which fail to express an active HPRT
enzyme are unable to grow in media containing HATG, but are
resistant to analogues such as 6-thioguanine, etc. (Littlefield
(1964) Science, 145: 709-710). Cells expressing active thymidine
kinase are able to grow in media containing HAT, but are unable to
grow in media containing nucleoside analogues such as
bromo-deoxyuridine (Giphart--Gassler et al. (1989) Mutat. Res. 214:
223-232). Cells containing an active HSV-tk gene are incapable of
growing in the presence of gangcylovir or similar agents.
[0306] The detectable marker gene may also be any gene that can
compensate for a recognizable cellular deficiency. Thus, for
example, the gene for HPRT could be used as the detectable marker
gene sequence when employing cells lacking HPRT activity. This
agent is an example of agents may be used to select mutant cells,
or to "negatively select" for cells which have regained normal
function.
[0307] In preferred embodiments, the chimeric or transgenic animal
cells of the present invention are prepared by introducing one or
more DNA molecules into a precursor pluripotent cell, most
preferably an ES cell, or equivalent (Robertson (1989) pages 39-44
In: Current communications in Molecular Biology, Capecchi, M. R.
(ed.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y.--The term
"precursor" is intended to denote only that the pluripotent cell is
a precursor to the desired ("transfected") pluripotent cell which
is prepared in accordance with the teachings of the present
invention. The pluripotent (precursor or transfected) cell may be
cultured in vivo, in a manner known in the art (Evans et al.,
(1981) Nature 292: 154-156) to form a chimeric or transgenic
animal. The transfected cell, and the cells of the embryo that it
forms upon introduction into the uterus of a female are herein
referred to respectively, as "embryonic stage" ancestors of the
cells and animals of the present invention.
[0308] Any ES cell may be used in accordance with the methods
described herein. In certain embodiments, it is, however, preferred
to use primary isolates of ES cells. Such isolates may be obtained
directly from embryos such as the CCE cell line disclosed by
Robertson, E. J., In: Current Communications in Molecular Biology,
Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring
Harbor, N.Y. (1989), pp. 39-44), or from the clonal isolation of ES
cells from the CCE cell line (Schwartzberg et al. (1989) Science
212: 799-803). Such clonal isolation may be accomplished according
to the method of Robertson (1987) In: Teratocarcinomas and
Embryonic Stem Cells: A Practical Approach, E. J. Robertson, Ed.,
IRL Press, Oxford. The purpose of such clonal propagation is to
obtain ES cells that have a greater efficiency for differentiating
into an animal. Clonally selected ES cells are approximately
10-fold more effective in producing transgenic animals than the
progenitor cell line CCE. An example of ES cell lines which have
been clonally derived from embryos are the ES cell lines, AB1
(hprt+) or AB2.1 (hprt-).
[0309] The ES cells are preferably cultured on stromal cells (such
as STO cells (especially SNL76/7 STO cells) and/or primary
embryonic G418 R fibroblast cells) as described by Robertson,
supra. Methods for the production and analysis of chimeric mice are
well known to those of skill in the art (see, e.g., Bradley (1987)
pages 113-151 In: Teratocarcinomas and Embryonic Stem Cells; A
Practical Approach, E. J. Robertson, ed., IRL Press, Oxford). The
stromal (and/or fibroblast) cells serve to eliminate the clonal
overgrowth of abnormal ES cells. Most preferably, the cells are
cultured in the presence of leukocyte inhibitory factor ("lif")
(Gough et al. (1989) Reprod. Fertil., 1: 281-288; Yamamori et al.
(1989) Science, 246: 1412-1416). Since the gene encoding lif has
been cloned (Gough, et al. supra.), it is especially preferred to
transform stromal cells with this gene, by means known in the art,
and to then culture the ES cells on transformed stromal cells that
secrete lif into the culture medium.
[0310] ES cell lines may be derived or isolated from any species
(for example, chicken, fish, etc.), although cells derived or
isolated from mammals such as rodents, rabbits, sheep, goats, pigs,
cattle, primates and humans are preferred. Cells derived from
rodents (i.e., mouse, rat, hamster, etc.) are particularly
preferred.
[0311] In fact, ES cell lines have been derived for mice and pigs
as well as other animals (see, e.g., Robertson, Embryo-Derived Stem
Cell Lines. In: Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach (E. J. Robertson, ed.), IRL Press, Oxford
(1987); PCT Publication No. WO/90/03432; PCT Publication No.
94/26884. Generally these cells lines must be propagated in a
medium containing a differentiation-inhibiting factor (DIF) to
prevent spontaneous differentiation and loss of mitotic capability.
Leukemia Inhibitory Factor (LIF) is particularly useful as a DIF.
Other DIFs useful for prevention of ES cell differentiation
include, without limitation, Oncostatin M (Gearing and Bruce (1992)
The New Biologist 4: 61-65), interleukin 6 (IL-6) with soluble IL-6
receptor (sIL-6R) (Taga et al. (1989) Cell 58: 573-581), and
ciliary neurotropic factor (CNTF) (Conover et al. (1993)
Development 19: 559-565). Other known cytokines may also function
as appropriate DIFs, alone or in combination with other DIFs.
[0312] As a useful advance in maintenance of ES cells in an
undifferentiated state, a novel variant of LIF (T-LIF) has been
identified (see U.S. Pat. No. 5,849,991). In contrast to the
previously identified forms of LIF which are extracellular, T-LIF
is intracellularly localized. The transcript was cloned from murine
ES cells using the RACE technique (Frohman et al. (1988) Proc.
Natl. Acad. Sci., USA, 85: 8998-9002) and subjected to sequence
analysis. Analysis of the obtained nucleic acid sequence and
deduced amino acid sequence indicates that T-LIF is a truncated
form of the LIF sequence previously reported in the literature.
Expression of the T-LIF nucleic acid in an appropriate host cell
yields a 17 kD protein that is unglycosylated. This protein is
useful for inhibiting differentiation of murine ES cells in
culture.
[0313] Production of the knockout animals of this invention is not
dependent on the availability of ES cells. In various embodiments,
knockout animals of this invention can be produced using methods of
somatic cell nuclear transfer. In preferred embodiments using such
an approach, a somatic cell is obtained from the species in which
the Idol gene is to be knocked out. The cell is transfected with a
construct that introduces a disruption in the Idol gene (e.g., via
heterologous recombination) as described herein. Cells harboring a
knocked-out Idol are selected as described herein. The nucleus of
such cells harboring the knockout is then placed in an unfertilized
enucleated egg (e.g., eggs from which the natural nuclei have been
removed by microsurgery). Once the transfer is complete, the
recipient eggs contain a complete set of genes, just as they would
if they had been fertilized by sperm. The eggs are then cultured
for a period before being implanted into a host mammal (of the same
species that provided the egg) where they are carried to term,
culminating in the birth of a transgenic animal comprising a
nucleic acid construct containing one or more disrupted Idol
genes
[0314] The production of viable cloned mammals following nuclear
transfer of cultured somatic cells has been reported for a wide
variety of species including, but not limited to frogs (McKinnell
(1962) J. Hered. 53, 199-207), calves (Kato et al. (1998) Science
262: 2095-2098), sheep (Campbell et al. (1996) Nature 380: 64-66),
mice (Wakayamaand Yanagimachi (1999) Nat. Genet. 22: 127-128),
goats (Baguisi et al. (1999) Nat. Biotechnol. 17: 456-461), monkeys
(Meng et al. (1997) Biol. Reprod. 57: 454-459), and pigs (Bishop et
al. (2000) Nature Biotechnology 18: 1055-1059). Nuclear transfer
methods have also been used to produce clones of transgenic
animals. Thus, for example, the production of transgenic goats
carrying the human antithrombin III gene by somatic cell nuclear
transfer has been reported (Baguisi et al. (1999) Nature
Biotechnology 17: 456-461).
[0315] Using methods of nuclear transfer as describe in these and
other references, cell nuclei derived from differentiated fetal or
adult, mammalian cells are transplanted into enucleated mammalian
oocytes of the same species as the donor nuclei. The nuclei are
reprogrammed to direct the development of cloned embryos, which can
then be transferred into recipient females to produce fetuses and
offspring, or used to produce cultured inner cell mass (CICM)
cells. The cloned embryos can also be combined with fertilized
embryos to produce chimeric embryos, fetuses and/or offspring.
[0316] Somatic cell nuclear transfer also allows simplification of
transgenic procedures by working with a differentiated cell source
that can be clonally propagated. This eliminates the need to
maintain the cells in an undifferentiated state, thus, genetic
modifications, both random integration and gene targeting, are more
easily accomplished. Also by combining nuclear transfer with the
ability to modify and select for these cells in vitro, this
procedure is more efficient than previous transgenic embryo
techniques.
[0317] Nuclear transfer techniques or nuclear transplantation
techniques are known in the literature. See, in particular,
Campbell et al. (1995) Theriogenology, 43:181; Collas et al. (1994)
Mol. Report. Dev., 38:264-267; Keefer et al. (1994) Biol. Reprod.,
50:935-939; Sims et al. (1993) Proc. Natl. Acad. Sci., USA,
90:6143-6147; WO 94/26884; WO 94/24274, WO 90/03432, U.S. Pat. Nos.
5,945,577, 4,944,384, 5,057,420 and the like.
[0318] Having shown that disruption of the Idol gene produces
Idol-deficient animals that are viable, one of skill will recognize
that there are a wide number of animals including natural and
transgenic animals that have other desirable phenotypes and that
can be used to practice the invention. Preferred animals are
mammals including, but not limited to, rodents (e.g, murines),
equines, bovines, porcines, lagomorphs, felines, canines, caprines,
ovines, non-human primates, and the like.
[0319] Zygotes or ES cells from the Idol knockouts of this
invention can be used as embryonic target cells for introduction of
other heterologous genes or knockout constructs. Alternatively
somatic cells can be used as targets for the introduction of
various heterologous expression cassettes or knockout
constructs.
[0320] In other embodiments, the knockout animals of this invention
can be can be cross-bred with other animals exhibiting various
natural or induced pathologies. In various embodiments, the
knockout animals of this invention are crossed with animals having
one or more knockouts other than the Idol knockout.
[0321] In certain preferred embodiments, a transgenic non-human
animal is bred that that includes a deficiency in Idol expression
(e.g., a heterozygous or homozygous Idol knockout) and a deficiency
in a second recombinantly disrupted gene.
V. Kits
[0322] In certain embodiments kits are provided for the treatment
methods and/or screening methods described herein (e.g., to inhibit
LDL receptor degradation and/or promote LDL uptake in a mammal,
and/or mitigate one or more symptoms of hypercholesterolemia, or to
screen for agents that inhibit Mylip expression and/or activity).
"Therapeutic" kits typically include a container containing one or
more Mylip inhibitors (e.g., siRNA, shRNA, etc.). Such kits can,
optionally include instruments for formulating or administering the
agent(s). Screening kits can include any of the reagents for
performing the screening assays described herein. In certain
embodiments, the kits comprise a construct containing a reporter
gene whose expression is regulated by Mylip or cell(s) comprising
such a construct. In addition the kits typically include
instructional materials disclosing means of use of the inhibitors
to slow aging, or instructional materials describing how to screen
for agents that slow aging. The kits can additionally include
buffers and other reagents routinely used for the practice of a
particular method. Such kits and appropriate contents are well
known to those of skill in the art.
[0323] While the instructional materials typically comprise written
or printed materials they are not limited to such. Any medium
capable of storing such instructions and communicating them to an
end user is contemplated by this invention. Such media include, but
are not limited to electronic storage media (e.g., magnetic discs,
tapes, cartridges, chips), optical media (e.g., CD ROM), and the
like. Such media may include addresses to internet sites that
provide such instructional materials.
EXAMPLES
[0324] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
[0325] LXR Regulates Cholesterol Uptake Through Mylip-Dependent
Ubiquitination of the LDL Receptor
[0326] The LDL receptor (LDLR) is a critical determinant of plasma
cholesterol levels. Here we show that the sterol-responsive nuclear
receptor LXR regulates LDLR-mediated lipoprotein uptake independent
of SREBPs. Ligand activation of LXR redistributes the LDLR from the
plasma membrane to an intracellular compartment and promotes its
degradation. This effect is accomplished through the
transcriptional induction of myosin light chain interacting protein
(Mylip), an E3 ubiquitin ligase. Enzymatically active Mylip
triggers ubiquitination of the LDLR on its cytoplasmic domain,
thereby targeting it for degradation. Mylip knockdown inhibits LDLR
degradation and promotes LDL uptake. Conversely, expression of
Mylip in mouse liver dramatically reduces LDLR protein and elevates
plasma LDL levels. The LXR-Mylip-LDLR axis defines a previously
unrecognized pathway for sterol regulation of LDL metabolism and an
enzyme target for the treatment of hypercholesterolemia.
Introduction.
[0327] The LDL receptor (LDLR) is central to the maintenance of
plasma cholesterol levels (Russell et al. (1984) Cell 37: 577).
Mutations in this receptor are the leading cause of autosomal
dominant hypercholesterolemia (ADH), characterized by defective
hepatic LDL uptake, elevated plasma cholesterol levels, and
increased risk of cardiovascular disease (. Tolleshaug et al.
(1983) Cell 32: 941; Brown and Goldstein (1986) Science 232: 34).
In line with its pivotal role in cholesterol homeostasis,
expression of the LDLR is tightly regulated. Transcription of the
LDLR gene is coupled to cellular cholesterol levels through the
action of the sterol response element binding protein (SREBP)
transcription factors (Yokoyama et al. (1993) Cell 75: 187; Hua et
al. (1993) Proc. Natl. Acad. Sci., USA, 90: 11603). Enhanced
processing of SREBPs to their mature forms when cellular sterol
levels decline leads to increased cholesterol biosynthesis and
enhanced LDLR transcription (Goldstein et al. (2006) Cell 124: 35).
Posttranscriptional regulation of LDLR expression is also a major
determinant of lipoprotein metabolism. Genetic studies have
identified mutations in the genes encoding the LDLR adaptor protein
1 (LDLRAP1/ARH) (Garcia et al. (2001) Science 292: 1394; Cohen et
al. (2003) Curr Opin Lipidol 14: 121) and the SREBP target gene
proprotein convertase subtilisin/kexin 9 (PCSK9) that result in
altered stability, endocytosis, or trafficking of the LDLR
(Abifadel et al. (2003) Nat Genet 34: 154; Cohen et al. (2005) Nat
Genet 37: 161; Seidah et al. (2003) Proc. Natl. Acad. Sci., USA,
100: 928; Maxwell et al. Proc. Natl. Acad. Sci., USA, 101: 7100;
Park et al. (2004) J Biol Chem 279: 50630).
[0328] The Liver X Receptors (LXRs) provide a complementary pathway
for the transcriptional control of cholesterol metabolism. LXRa
(NR1H3) and LXR(3 (NR1H2) are sterol-dependent nuclear receptors
activated in response to cellular cholesterol excess (Zelcer and
Tontonoz (2006) J Clin Invest 116: 607). LXRs target genes such as
ABCA1 and ABCG1 promote the efflux of cellular cholesterol and help
to maintain whole-body sterol homeostasis (Repa et al. (2000)
Science 289: 1524; Kennedy et al. (2005) Cell Metab 1: 121). Mice
lacking LXRs develop marked accumulation of sterols in their
tissues and accelerated atherosclerosis, whereas synthetic LXR
agonists promote reverse cholesterol transport and protect against
atherosclerosis (. Peet et al. (1998) Cell 93: 693; Tangirala et
al. (2002) Proc. Natl. Acad. Sci., USA, 99: 11896; Joseph et al.
(2002) Proc. Natl. Acad. Sci., USA, 99: 7604). The coordinated
regulation of intracellular sterol levels by the LXR and SREBP
signaling pathways led us to hypothesize that LXRs may act to limit
cholesterol uptake in addition to promoting cholesterol efflux.
[0329] We initially tested the ability of LXRs to modulate LDL
uptake in cultured cells. Treatment of HepG2 cells with either of
two structurally-unrelated synthetic LXR ligands (GW3695 or T1317)
decreased binding and uptake of bodipy labeled-LDL (FIG. 1A). As
the LDLR is the major route of LDL uptake, we tested the effects of
LXR agonists on LDLR expression. Activation of LXR decreased LDLR
protein expression in HepG2 cells independent of cellular sterol
levels (FIG. 1B). The LDLR receptor runs as a doublet on western
blots, with the ratio of the upper and lower bands varying by cell
type. The upper band (m) is the mature glycosylated form found in
the plasma membrane, while the lower band corresponds to an
immature precursor (p). The effect of LXR was most prominent on the
mature LDLR. Remarkably, this reduction occurred despite a slight
increase in LDLR transcript, likely secondary to induction of
SREBP-1c expression by LXR (FIG. 1C). The ability of LXRs to
decrease LDLR proteins levels was also apparent in human SV589
fibroblasts that have been used extensively to delineate SREBP
processing (FIG. 1D) (Adams et al. (2004) J Biol Chem 279: 52772)
Reduction in LDLR protein by LXR ligands in SV589 cells was not
paralleled by changes in HMGCoA reductase expression (FIG. 1D) and
could not be attributed to a decrease in LDLR mRNA (FIG. 5A).
[0330] To determine whether the effect of the synthetic ligands on
LDLR was LXR-dependent, we used immortalized
Lxr.alpha..beta..sup.(-/-) mouse embryonic fibroblasts (MEFs)
stably expressing LXRa or vector control. Oxysterol LXR ligands
block SREBP processing in addition to activating LXR, and therefore
22(R)-hydroxycholesterol decreased LDLR protein in the absence or
presence of LXRs (FIG. 1E). By contrast, the synthetic ligand
GW3965 decreased LDLR only in cells expressing LXRa. To investigate
the link between endogenous LXR ligands and LDLR expression we
utilized an adenovirus expressing oxysterol sulfotransferase
(Sult2b1). Depletion of oxysterol agonists by Sult2b1 in SV589
cells led to increased LDLR protein, and this effect was reversed
by synthetic ligand (FIG. 1F). To further delineate the role of
LXRs in regulating LDLR expression, we tested the effect of LXR
agonists on LDLR produced from a transfected vector not subject to
endogenous SREBP regulation. In HepG2 cells stably expressing an
LDLR-GFP fusion protein, LDLR-GFP expression was visualized
primarily on the plasma membrane (FIG. 1G). Ligand activation of
LXR decreased overall LDLR-GFP expression and redistributed the
LDLR from the plasma membrane to intracellular compartments (FIG.
1G).
[0331] The above results indicate that LXR signaling modulates LDLR
expression independent of the SREBP pathway. We reasoned that an
LXR target gene must underlie this effect, possibly one that
promotes degradation of the LDLR. Using transcriptional profiling
we identified a potential mediator: the myosin light chain
interacting protein (Mylip) (data not shown). Mylip is a member of
the Ezrin/Radixin/Moesin (ERM) family of proteins that mediates
interactions between proteins and membrane structures (Bretscher et
al. (2992) Nat Rev Mol Cell Biol 3: 586). Distinguishing it from
other members of this family, Mylip contains a C-terminal RING
domain and acts as an E3-ubiquitin ligase (Olsson et al. (1999) J
Biol Chem 274: 36288; Bornhauser et al. (2003) FEBS Lett 553: 195).
LXR agonists strongly induced Mylip expression in cell lines and
primary cells of human and rodent origin in an LXR-dependent manner
(FIGS. 2A and 2B). Furthermore, Mylip expression was induced in
vivo following administration of GW3965 to mice (FIG. 2C).
[0332] LXR regulation of Mylip was not sensitive to the ribosomal
poison cycloheximide, suggesting that it is a direct
transcriptional effect (FIG. 6A). It also could not be secondary to
induction of SREBP-1c, because oxysterols that block SREBP
processing still induced Mylip expression (FIG. 6B). LXRs activate
target genes by binding to consensus elements (LXREs) in their
promoters. We identified an LXRE approximately 2.5 kb upstream of
the mouse Mylip translation start site (FIG. 6C) and generated a
reporter construct encompassing this region. Activation by LXRa and
GW3965 resulted in a .about.4-fold increase in reporter activity
that was largely abolished in the absence of a functional LXRE
(FIG. 6D). EMSA analysis showed that LXR/RXR heterodimers bound to
wild type but not mutant versions of the Mylip LXRE and that
binding could be competed by an excess of unlabeled Mylip LXRE
(FIG. 6E).
[0333] Ubiquitination plays an important role in modulating protein
expression. Given that Mylip is a putative E3 ubiquitin ligase, we
hypothesized that Mylip induction might underlie the ability of
LXRs to inhibit LDLR expression. Employing a co-transfection system
in HEK293T cells we found that both human and mouse Mylip potently
reduced the expression of LDLR-GFP in a dose-dependent manner (FIG.
2D). Moreover, consistent with the effect observed with LXR
agonists, Mylip expression redistributed LDLR-GFP expression from
the plasma membrane to an intracellular compartment (FIG. 2E and
FIG. 7). By contrast, Mylip carrying a point mutation (C387A) in
the catalytic RING domain had no effect on LDLR expression or
localization. Also, in line with the effects of LXR ligand (FIG.
1), the effects of Mylip were most prominent on the fully
glycosylated mature LDLR form (FIGS. 2D and 2F). Notably, Mylip
expression was greatly enhanced when the RING domain was mutated
raising the possibility that Mylip might catalyze its own
degradation.
[0334] The effect of Mylip on membrane protein expression appears
to be selective for the LDLR. Levels of transfected LRP1-GFP or
APP-GFP proteins, both of which contain NPXY motifs and undergo
regulated endocytosis similar to the LDLR, were unaffected by Mylip
expression (FIG. 2F). Mylip also did not influence expression of
ABCA1-GFP, endogenous transferrin receptor (TFRC), or endogenous
myosin regulatory light chain (MRLC). Importantly, regulation of
LDLR receptor by Mylip was observed in multiple cell types,
including primary hepatocytes (FIG. 2G), as well as HepG2 cells,
macrophages, and fibroblasts (data not shown).
[0335] We next sought to determine whether Mylip expression could
recapitulate the functional effects of LXR activation on LDL
uptake. Indeed, expression of Mylip in MEFs from an adenoviral
vector dramatically reduced LDL binding and uptake (FIG. 2H).
Conversely, introduction of two independent shRNAs targeting mouse
Mylip into MEFs increased LDLR protein without affecting LDLR mRNA
(FIG. 2I and FIG. 8A). Importantly, the magnitude of the change in
LDLR expression we observed following Mylip knockdown was
comparable to that observed following inhibition of endogenous
sterol production by statins (FIG. 8B). This observation suggests
that modulation of Mylip activity is a physiological mechanism for
regulating LDLR abundance.
[0336] In support of this idea, introduction of either Mylip shRNA
construct into MEFs increased LDL uptake (FIG. 2J). Finally, the
ability of an LXR ligand to reduce LDLR protein levels was
substantially diminished when Mylip expression was knocked down,
directly implicating Mylip in LXR-dependent regulation of LDLR
expression (FIG. 2K).
[0337] Having identified Mylip as a regulator of LDLR expression,
we endeavored to determine its mechanism of action. The data above
suggest that a post-transcriptional event is involved. Pulse-chase
labeling studies showed that Mylip did not block LDLR message
translation or appearance of the immature protein, but it markedly
reduced abundance of the mature form, pointing to a
post-translational degradation event (FIG. 3A). The fact that
mutation of the RING domain inactivates Mylip (FIG. 2E) implicates
the E3 ligase activity in its mechanism of action. But what is the
protein target for Mylip-directed ubiquitination? The most
straightforward explanation for our data is that Mylip triggers
ubiquitination of the LDLR itself, thereby marking it for
degradation. The LDLR has not previously been shown to be
ubiquitinated; however, to our knowledge this possibility has not
been examined in contexts where LXR signaling or Mylip were
specifically altered. We found that although basal
polyubiquitination of the LDLR in 293T cells was minimal, it was
dramatically enhanced by expression of active but not mutant Mylip
(FIG. 3B).
[0338] Next, we determined the structural requirements for LDLR
degradation by Mylip. The 50 amino acid cytoplasmic tail of the
LDLR links it to the endocytic machinery and cytoplasmic adapter
proteins (Gotthardt et al. (2000) J Biol Chem 275: 25616). Mylip
action on the LDLR was independent of the NPXY endocytosis motif in
the cytoplasmic tail, because Mylip effectively reduced expression
of an LDLR mutant (Y18A) defective in endocytosis (FIG. 9A)
(Hunziker et al. (1991) Cell 66: 907). However, Mylip had no effect
on an LDLR receptor lacking the entire intracellular domain (FIG.
3C). Typically, ubiquitination occurs on lysine residues, however,
some membrane receptors have recently been demonstrated to be
ubiquitinated on cysteine residues (Cadwell and Coscoy (2005)
Science 309: 127). The LDLR intracellular domain contains 3 highly
conserved lysines and one cysteine (FIG. 3D). We generated a panel
of single and compound mutants of these LDLR residues and tested
their effects on Mylip action. Remarkably, single mutations of any
of these residues, or even loss of all three lysine residue, did
not prevent Mylip from degrading the LDLR (FIG. 3E). However,
superimposing the cysteine mutation on constructs containing two or
three mutated lysines rendered the LDLR insensitive to degradation.
To further narrow down the residues targeted by Mylip, we generated
additional combined lysine and cysteine mutations (FIG. 3F). This
analysis revealed that either an intact K20 or an intact C29 was
required for Mylip mediated degradation. Only when these two
residues were mutated together was Mylip action substantially
blocked. Combined mutations of any of the three lysines with a
mutation in I58, adjacent to C59, had no effect (FIG. 9B). Notably,
both K20 and C29 are highly conserved among species, suggesting
that the Mylip regulatory circuit may also be conserved (FIG. 3D).
Finally, not only did combined mutation of the K20 and C29 residues
block LDLR degradation by Mylip, it also blocked ubiquitination
(FIG. 3G). Interestingly, the proteosome blocker MG132, despite
greatly stabilizing Mylip expression, did not increase the
Mylip-dependent level of LDLR ubiquitination. This observation is
consistent with previous reports suggesting that degradation of the
LDLR does not occur in the proteosome (FIG. 3G). These results
strongly support the hypothesis that reduction of LDLR protein by
Mylip involves directed ubiquitination of the cytoplasmic
domain.
[0339] To study the physiological function of Mylip in vivo we
transduced mice with adenoviral vectors encoding LacZ or mouse
Mylip. Analysis of serum AST and ALT levels showed no evidence of
hepatotoxicity due to viral infection (data not shown).
[0340] Remarkably, Mylip expression resulted in a striking increase
in plasma levels of total and unesterified cholesterol (FIG. 4A).
Levels of triglycerides, free fatty acids and glucose were not
altered significantly. Fractionation of plasma revealed that Mylip
expression caused a phenotype reminiscent of Ldlr.sup.(-/-) mice,
characterized by a dramatic shift in the lipoprotein profile and
the appearance of an LDL peak not present in the control mice (FIG.
4B). A slight increase in triglyceride content in the
LDL-containing fractions was also observed (FIG. 4C). Western
blotting confirmed the presence of apoB in this peak (FIG. 4D).
Consistent with our in vitro results, hepatic expression of LXR and
SREBP-2 target genes was not affected by Mylip expression (FIG.
4E). Rather, Mylip strongly reduced hepatic LDLR protein levels,
providing a straightforward explanation for the appearance of an
LDL fraction (FIG. 4F). By contrast, transferrin receptor
expression was not altered by Mylip. An independent in vivo
experiment showing virtually identical results is presented in
FIGS. 10A-10C. Finally, Mylip adenovirus had no effect on plasma
cholesterol levels or lipoprotein profiles or cholesterol levels of
Ldlr.sup.(-/-) mice, unequivocally demonstrating that Mylip action
is dependent on the LDLR (FIG. 4G, FIG. 10D, and FIG. 10E).
[0341] We have shown here that the sterol-sensitive nuclear
receptor LXR regulates LDLR-dependent cholesterol uptake through a
pathway independent of and complementary to the SREBPs. Activation
of LXR induces expression of Mylip, which in turn catalyzes the
ubiquitination of the LDLR, thereby targeting it for degradation.
Our results offer a mechanistic explanation for an
under-appreciated observation made two decades ago by Witztum and
colleagues, who documented regulation of an ectopically-expressed
LDLR by sterols (Sharkey et al. (1990) J Lipid Res 31: 2167). The
physiological relevance of the LXR-Mylip-LDLR pathway is clear from
our demonstrations that blocking endogenous Mylip expression
promotes LDL uptake and that mice overexpressing Mylip in their
livers phenocopy LDLR-/- mice.
[0342] Mechanistically we have established that Mylip triggers
ubiquitination of the LDLR on K20 and C29 and that these residues
are required for Mylip-dependent degradation. However, further
study will be required to fully define the degradation pathway.
Inhibition of the proteosome does not lead to the accumulation of
poly-ubiquitinated LDLR, even when active Mylip is present. This
suggests that Mylip-driven LDLR degradation is non-proteosomal,
similar to what has been reported for Pcsk9 (Maxwell et al. (2005)
Proc. Natl. Acad. Sci., USA, 102: 2069). Ubiquitination of membrane
receptors can serve as a trafficking signal to direct proteins to
specific organelles (Bonifacino and Traub (2003) Annu Rev Biochem
72: 395). It is therefore tempting to postulate that Mylip may act
along the route LDLR traffics within the cell to direct it to a
specific organelle(s) where it is degraded.
[0343] Another important issue to be resolved is the potential
relationship between the Pcsk9 and Mylip pathways. Pcsk9 is a
secreted protein induced by SREBP activation that may serve as a
feedback inhibitor of SREBP action on the LDLR (Park et al. (2004)
J Biol Chem 279: 50630; Maxwell et al. (2005) Proc. Natl. Acad.
Sci., USA, 102: 2069). Pcsk9 binds to the LDLR extracellular
ligand-binding domain, causing its removal from the cell surface
and degradation. As Pcsk9 is a secreted protein and Mylip is
cytoplasmic, it is unlikely that these proteins interact directly.
However, despite extensive studies of Pcsk9-mediated LDLR
degradation, the cellular location and mechanism of degradation are
still unclear. An exciting but as yet unexplored possibility is
that Mylip may act downstream of Pcsk9 in the same pathway to
regulate LDLR expression.
[0344] Our identification of the Mylip-LDLR pathway fills a
significant gap in our understanding of how LXRs control
cholesterol homeostasis. The ability of LXRs to respond to excess
cellular cholesterol by promoting efflux through ABC transporters
has been extensively documented (Zelcer and Tontonoz (2006) J Clin
Invest 116: 607). It makes intuitive sense that a sterol-activated
transcription factor might also be employed to limit LDL
cholesterol uptake, but this has not been described previously. The
LXR-Mylip-LDLR pathway provides such a mechanism. Our results
further illustrate how the two major sterol-regulated
transcriptional factors, SREBP and LXR, act in a complementary and
coordinated fashion to maintain cholesterol homeostasis. In fact,
it may be very difficult to separate the components of these
pathways in physiological contexts. Most endogenous oxysterol
ligands of LXR inhibit SREBP processing. SREBP activation promotes
LDLR expression, but also induces expression of the inhibitor
Pcsk9. Systemic activation of LXR not only induces Mylip, but also
promotes cholesterol efflux, induces SREBP-1c expression,
stimulates bile acid synthesis, and blocks intestinal cholesterol
absorption. Interestingly, LXR ligands have been reported to raise
LDL levels in hamsters and primates and Mylip may be a contributor
to this effect (Groot et al. (2005) J Lipid Res 46: 2182).
[0345] The LDL pathway is a validated target for cardiovascular
therapy. Statin drugs act primarily by increasing hepatic
expression of the LDLR, leading to increased LDL clearance. The
possibility that pharmacologic targeting of the Mylip pathway
provides a complementary strategy for increasing LDLR expression is
intriguing. Indeed, Pcsk9 is being actively investigated as such a
complementary target. In support of this idea, we have shown that
knockdown of endogenous Mylip expression using siRNAs increases
both LDLR expression and LDL uptake. Moreover, the fact that Mylip
is an enzyme potentially opens the door for the development of
small molecule inhibitors. The discovery that the enzymatic
activity of Pcsk9 was not required for its action was disappointing
from a drug development standpoint. By contrast, Mylip's E3 ligase
activity appears critical for its action on the LDLR.
Abbreviations
[0346] Liver X Receptors; LXRs
[0347] LDL receptor; LDLR
[0348] Myosin Light Chain Interacting Protein; Mylip
[0349] LXR response element; LXRE
[0350] mouse embryonic fibroblasts; MEFs
Materials & Methods
[0351] Reagents
[0352] The synthetic LXR ligands GW3965 and T0901317 were provided
by T. Wilson (GlaxoSmithKline). MG132, Mevalonic acid, and
22R-hydroxycholesterol were from Sigma-Aldrich. Simvastatin sodium
salt was from Calbiochem. BODIPY-LDL was purchased from Molecular
Probes and fetal bovine lipoprotein deficient serum (LPDS) from
Intracell.
[0353] Plasmids and Expression Constructs
[0354] The pEGFP-N-3-hLDLR and its Y18A variant were a kind gift
from Dr. A. Gonzalez (School of Medicine, Cassila, Chile) (Cancino
et al. (2007) Mol Biol Cell 18: 4872). The non-tagged pCB6-hLDLR
and its intracellular deletion mutant (pCB6-.DELTA.hLDLR) were a
kind gift from Dr. K. Matter (UCL, England) (Matter et al. (1992)
Cell 71: 741). Site directed mutagenesis was used to introduce
mutations in pCB6-hLDLR with the Quickchange multi-site mutagenesis
kit (Stratagene). pEGFP-N-1-hABCA1 was a kind gift of Dr. K. Ueda
(Kyoto University, Japan) (Tanaka et al. (2003) J Biol Chem 278:
8815). The pEGFP-N-1-hAPP and pEGFP-N-1-light-chain-hLRP1 were a
kind gift from Dr. B. Hyman (Harvard Medical School, USA)
(Kinoshita et al. (2002) J Neurochem 82: 839). The
pcDNA3.1-(HA-Ubiquitin).sub.6 was a kind gift from Dr. J.
Wohlschlegel (UCLA, USA). The full-length human and mouse MYLIP
cDNAs were amplified by PCR from IMAGE clone 3638617 and IMAGE
clone 3964381, respectively, and cloned into the gateway entry
plasmid pENTR1A (Invitrogen). The mMylip cDNA in IMAGE clone
3964381 contains 2 nucleotide changes from the mMylip reference
sequence (NM.sub.--153789) that result in a P113A amino acid
change. These two nucleotides were reverted to conform to the
reference sequence using site directed mutagenesis. Additionally, a
C387A mutation in the RING domain of both human and mouse MYLIP was
introduced by site directed mutagenesis. To generate mammalian
expression constructs for MYLIP we used LR recombination between
MYLIP-containing pENTR1A and pDEST47 (Invitrogen). Restriction
digest analysis and DNA sequencing were used to verify the
correctness of all the constructs used in this study.
[0355] Mylip Promotor Analysis
[0356] A 10 Kb promotor region upstream of the mMylip transcription
initiation site was analyzed for the presence of LXR binding
elements. A consensus DR4 site was identified 2454 upstream of the
transcription initiation site. Olignoucleotides encompassing this
element were designed, annealed and tested for binding to LXR/RXR
heterodimers in a standard EMSA assay. A 2.5 kB promotor fragment
upstream of the transcription initiation site containing the
identified LXRE was amplified by PCR from BAC clone RP23-17N24
using mMylip-prom-FWD GCTAGCCCTACTTAACCTACAATGACCT (SEQ ID NO:20)
or mMylip-prom-.DELTA.FWD GCTAGCTTCTGATGCTTCTACCTCTATC (SEQ ID
NO:21) with the common antisense primer mMylip-prom-REV
GAGCTCAGTTCCCGGGAGCTACACG (SEQ ID NO:22). Amplified fragments were
cloned as SacI/XhoI fragments into pGL3 basic (Promega). Promotor
analysis was done as previously described (Joseph et al. (2002) J
Biol Chem 277: 11019).
[0357] Generation and Amplification of Adenoviral Particles
[0358] Ad-mMylip particles were generated by LR recombination of
pENTR1A-mMylip and pAd/CMV/V5-DEST (Invitrogen). To generate
Ad-shMylip virus we used the pAd-BLOCK-iT kit (Invitrogen)
following the manufacturer's instructions. Briefly,
oligonucleotides targeting 4 different regions of mMylip (see Table
2) were designed with proprietary software from Invitrogen and
cloned into pU6-ENTR. The resulting pU6-mMylip.sup.shRNA plasmids
were tested for their ability to inhibit mMylip expression in
transient transfection experiments in HEK293T cells. The two
constructs showing the greatest inhibition were LR recombined with
pAD/BLOCK-iT-DEST (Invitrogen) to generate pAd-shMylip#1 and
pAd-shMylip#2. Viruses used in this study were amplified, purified
and tittered by Viraquest.
TABLE-US-00002 TABLE 2 Sequences of oligonucleotides used for
constructing shRNA constructs. All sequences target mMylip SEQ
Name/Set Sequence (5'-3') ID NO Set 1: NZ#288-U6-1F
caccGGAGCAAAGGTGAGAGCTTAT ATAAGCTCTCACCTTT 23 GCTCC NZ#289-U6-1R
aaaaGGAGCAAAGGTGAGAGCTTAT ATAAGCTCTCACCTTT 24 GCTCC Set 2:
NZ#290-U6-2F caccGCCTTAAACTGAGGGTCAAGT ACTTGACCCTCAGTTT 25 AAGGC
NZ#291-U6-2R aaaaGCCTTAAACTGAGGGTCAAGT ACTTGACCCTCAGTTT 26 AAGGC
Set 3: NZ#292-U6-3F caccGGACAGCGAAGGACAGAAACT AGTTTCTGTCCTTCGC 27
TGTCC NZ#293-U6-3R aaaaGGACAGCGAAGGACAGAAACT AGTTTCTGTCCTTCGC 28
TGTCC Set 4: NZ#294-U6-4F caccGCATCGTGCTCCTGTTTAAGA
TCTTAAACAGGAGCAC 29 GATGC NZ#295-U6-4R aaaaGCATCGTGCTCCTGTTTAAGA
TCTTAAACAGGAGCAC 30 GATGC Lower case: adaptor sequences for U6
plasmid Normal: FWD complementary sequence Italics: shRNA loop
sequence Underline: REV complementary sequence
[0359] Cell Culture, Transfections and Adenoviral Infections
[0360] The cell lines HEK293T, HepG2, and SV589 were obtained from
ATCC. HEK293T were cultured in Optimem containing 2% FBS. HepG2 and
SV589 were cultured in DMEM supplemented with 10% FBS. immortalized
Lxr.alpha..beta..sup.(-/-) mouse embryonic fibroblasts (MEFs) were
obtained by immortalizing E13.5 MEFs with a SV40 Large-T antigen
retrovirus. To reconstitute mLxr.alpha. we transfected Phenix cells
with pBabehygro or pBabehygro-mLxra. The resulting supernatant
containing the corresponding retroviral particles was used to
infect immortalized MEFs. Cells were selected in 800 .mu.g/mL
Hygromycin and surviving cells were used. HepG2-LDLR-GFP cells were
generated by transfecting HepG2 cells with pEGFP-N-1-LDLR and
selecting cells with G418 (800 .mu.g/mL).
[0361] Primary hepatocytes were isolated and maintained as
previously reported (Pei et al. (2006) Nat Med 12: 1048). HEK293T
cells were transfected using lipofectamine-2000 (invitrogen).
Typically, 0.8.times.10.sup.6 cells were seeded in a 60 mm well and
transfected the following day with 2 .mu.g DNA. In experiments
testing the ability of Mylip to degrade other potential protein
targets a ratio of 3:1 (Mylip:target) was used. To infect MEFs and
SV589 with adenovirus cells were seeded (0.2.times.10.sup.6
cells/60 mm well) and infected the following day at an MOI of 300.
HepG2 cells were infected at an MOI of 80. Primary hepatocytes were
infected 4 hrs after plating at an MOI of 20. Infections were
allowed to proceed for 18 hrs after which culture medium was
replaced. Under these conditions more than 90% of the cells were
transduced as assessed by GFP immunofluorescence. Where indicated,
cells were subsequently sterol starved by incubating cells in DMEM
supplemented with 10% LPDS, 5 M Simvastatin, and 100 M Mevalonic
acid for an additional 18 hrs (sterol deficient medium).
[0362] LDL Uptake Assay
[0363] HepG2 cells were plated at density of 40,000 cells/well. On
the following day the cells were pre-treated for 8 hrs with DMSO,
1M GW3965, or 1 .mu.M T0901317 in DMEM supplemented with 10% FBS.
MEFs (DKO-mLxr.alpha.) were plated similarly and infected on the
following day with the indicated adenovirus (MOI 300) for 18 hrs.
Subsequently, cells were washed twice with PBS and incubated for an
additional 16 hrs in sterol deficient medium to induce expression
of the LDLR. In experiments with HepG2 cells the sterol deficient
medium further contained the LXR synthetic ligands indicated above.
Uptake was initiated by incubating cells in 200 .mu.l of serum
deficient medium containing 5 .mu.g/mL of BODIPY-LDL. For measuring
binding of BODIPY-LDL to cells, plates were placed on ice prior to
processing. Uptake of BODIPY-LDL was measured after a 30 minute
incubation period at 37.degree. C. At that point cells were washed
2.times. with PBS containing 0.2% BSA and lysed in 100 .mu.l RIPA
buffer supplemented with protease inhibitors. Lysates were
collected and cleared by centrifugation and 30 .mu.l of cleared
lysate was transferred into a 384 well plate and measured on a
Typhoon apparatus (Amersham) with filters set for BODIPY. The
protein concentration of the cleared lysates was determined and
results are presented as relative fluorescence normalized for
protein content.
[0364] RNA Isolation and Quantitative RT-PCR
[0365] Total RNA was isolated from cells and mouse tissues using
Trizol (Invitrogen). One microgram of total RNA was reverse
transcribed with random hexamers using iScript reverse
transcription reagents kit (Biorad). Sybergreen (Diagenode)
real-time quantitative PCR assays were performed using an Applied
Biosystems 7900HT sequence detector. Results show averages of
duplicate experiments normalized to 36B4. Sequences for qPCR
primers are shown in Table 3.
TABLE-US-00003 TABLE 3 qPCR oligonucleotide pairs. SEQ ID Name/Set
Species Sequence (5'-3') NO Human Set 1: NZ#179-hMYLIP-F human
cgaggactgcctcaacca 31 NZ#180-hMYLIP-R human
tgcagtccaaaatagtcaacttct 32 Human Set 2: NZ#284-hMYLIP new1-F human
ttcttcgtggagcctcatct 33 NZ#285-hMYLIP new1-R human
cctccttgatgtgcaagaaaa 34 Human Set 3: NZ#286-hMYLIP new2-F human
atgaggagctctgtgccaag 35 NZ#287-hMYLIP new2-R human
tccttatgttttgcaacaatgc 36 Mouse Set 1: bv-mMylip-458F mouse
tgtggagcctcatctcatctt 37 bv-mMylip-526R mouse
agggactctttaatgtgcaagaa 38 Mouse Set 2: NZ#197-mMylip(new1)-F mouse
cagctatgaggacctgtgtgag 39 NZ#198-mMylip(new1)-R mouse
tccttatgcttcgcaacgat 40 Mouse Set 3: NZ#199-mMylip(new2)-F mouse
aggagatcaactccaccttctg 41 NZ#200-mMylip(new2)-R mouse
atctgcagaccggacagg 42
[0366] Antibodies, Immunoblot Analysis and Immunoprecipitation
[0367] Total cell or tissue lysates were prepared in RIPA buffer
(150 mM NaCl, 1% NP-40, 0.1% Sodium Deoxycholate, 0.1% SDS, 100 mM
Tris-HCl, pH 7.4) supplemented with protease inhibitors (Roche
Molecular Biochemicals). Lysates were cleared by centrifugation at
4.degree. C. for 10 minutes at 10,000 g. Protein concentration of
the cleared lysates was determined using the Bradford assay
(Biorad) with BSA as reference. Samples (10-40 .mu.g) were
separated on NuPAGE Bis-Tris gels (Invitrogen) and transferred to
nitrocellulose. Membranes were probed with the following
antibodies: LDLR (Cayman chemical, 1:1000), ABCA1 (Novus, 1:1000),
Tubulin (Calbiochem, 1:10000), HMGCR (rabbit anti-HMGCR polysera
was a gift from Dr. Peter Edwards (Edwards et al. (1983) J Biol
Chem 258: 7272), UCLA, 1:1000), GFP (affinity purified rabbit
polyclonal anti-GFP was a gift from Dr. Mireille Riedinger, UCLA,
1:5000), TFRC (Zymed, 1:5000), MRLC (Cell Signaling, 1:1000) and
poly-ubiquitin (Biomol, 1:1000). Polysera against MYLIP was raised
in rabbits following immunization with a KLH-conjugated peptide
corresponding to amino acids 316-329 of human and mouse MYLIP
(Sigma-genosys) and used at a dilution of 1:1000. Appropriate
secondary HRP-conjugated antibodies (DAKO) were used and visualized
with chemiluminescence (ECL, Amersham). To immunoprecipitate
LDLR-GFP, lysates were prepared as above and pre-cleared by
incubation with Protein-A agarose beads (Santa Cruz) for 30
minutes. Subsequently, equal amounts of protein of cleared lysate
were incubated with anti-GFP polysera (1:1000) for 30 minutes prior
to addition of Protein-A agarose beads for an additional 16 hrs.
For HA immunoprecipitations, equal amounts of protein of cleared
lysates were incubated with EZ view red anti-HA affinity beads
(Sigma) for 16 hrs. Subsequently, beads were washed 4.times. with
RIPA buffer supplemented with protease inhibitors. All incubations
and washes were done at 4.degree. C. with rotation. Proteins were
eluted from the beads by boiling in 1.times. protein sample buffer
for 5 minutes.
[0368] Metabolic Labeling of Cells
[0369] HepG2-LDLR-GFP cells were infected with adenovirus as
indicated above for 18 hrs. Subsequently, cells were washed
2.times. with PBS and pulsed for 30 minutes with DMEM lacking
Methionine and Cysteine (MP-Biochemicals) supplemented with 200
.mu.Ci/well easy Tag express .sup.35S protein labeling mix (Perkin
Elmer). Cells were then washed 3.times. and chased in DMEM
containing 10% FBS and 100 .mu.g/mL Methionine and 500 .mu.g/mL
Cysteine for the indicated times. Preparation of cell lysates and
immunoprecipitation of LDLR-GFP was conducted as detailed
above.
[0370] Animal Experiments
[0371] C57BL/6 mice and C57BL/6 Ldlr.sup.(-/-) mice (Jackson
Laboratoryfed) were fed a standard chow diet and housed in a
temperature-controlled room under a 12-hour light/12-hour dark
cycle under pathogen-free conditions. For adenoviral infections,
age-matched (8-10 weeks old) male mice were injected with 1.5XE109
PFU by tail-vein injection. Mice were sacrificed 6 days later
following a 6 hr fast. At the time of sacrifice liver tissue and
blood was collected by cardiac-puncture and immediately frozen in
liquid nitrogen and stored at -80.degree. C. Liver tissue was
processed for isolation of RNA and protein as above. Plasma lipids
were determined as previously reported. Animal experiments were
conducted in accordance with the UCLA Animal Research
Committee.
[0372] Statistical Analysis
[0373] Real-time PCR data, LDL-uptake assays, and plasma lipid
parameters are expressed as mean.+-.standard deviation. Statistical
analysis was done with a two-tailed Student's t-test. A probability
value of p<0.05 was considered statistically significant.
Example 2
Further Studies
Generation and Validation of Idol-/- Cells as an Independent
Screening Method.
[0374] We have generated homozygous Idol-/- ES cells from our
targeted heterozygous ES clones through high dose G418 selection.
As shown in FIG. 12A, these cells completely lack expression of
Idol mRNA. Interestingly, basal and LXR-dependent expression of
LDLR protein is markedly altered in the genetic absence of Idol.
The Idol-/- ES cells express significantly higher levels of LDLR
protein (FIG. 12B).
[0375] We propose a screen for small molecules that affect the Idol
pathway based on the Idol-/- cells. WT and Idol-/- are screened for
response to candidate small molecules. The effect of Idol-specific
small molecules will be lost in the Idol-/- cells. This screening
method will be used in conjunction with the cell-based reporter
screens described previously.
Identification of the Idol-LDLR Recognition Sequence.
[0376] Our data indicate that the FERM domain interacts directly
with the cytoplasmic tail of the LDLR and provides target
recognition, while the RING domain is essential for E3 ligase
activity. Since Idol is the only protein in the mammalian genome
with both FERM and RING domains, this model provides an attractive
explanation for the specificity of Idol effects and can be used as
the basis to screen for inhibitors. The Idol FERM domain is
predicted to contain 4 distinct loops (F1, F2, F3 and F3a; FIG.
13). We have determined that both the FERM and RING domains are
required for Idol action on the LDLR (FIG. 14). In addition, the F1
loop of Idol is dispensable for LDLR degradation, but the F3a loop,
which is unique to Idol and not present other FERM domains, is
essential (FIG. 14).
[0377] Furthermore, as shown in FIG. 15, the two proteins most
closely related by sequence to the LDLR, the VLDLR and apoER2, are
both capable of being degraded by Idol in our cotransfection assay.
Several other members, including LRP-1, LRP-1b and the EGFR
receptor are not. Based on these results, we can conclude that
neither the NPVY internalization motif, nor the presence of the
LDLR lysine or cysteine ubiquitination sites are sufficient to
confer Idol recognition. Rather, the residues conserved between
LDLR, VLDLR and apoER2 are important for Idol recognition. We have
further determined that the FERM domain of Idol is critical for
interaction with the LDLR. Disruption of Idol FERM domain
interaction with these LDLR residues using a small molecule would
inactivate the Idol-LDLR pathway.
[0378] The Idol-LDLR recognition sequence can be used as the basis
for a screen aimed at identifying small molecules that specifically
disrupted Idol-LDLR interaction by targeting this region of the
LDLR.
Use of Idol Transgenic and Knockout Mice as the Basis of Screens
for Small Molecules Targeting the Idol Pathway.
[0379] We have generated transgenic (Tg) mice that express human
Idol from the liver-specific albumen promoter. We have established
two independent lines and confirmed that these mice express human
Idol specifically in their livers (FIG. 16). Quantitative realtime
PCR has established that line A expresses approximately 2.times.
more Idol mRNA compared to C57B1/6 mice and line B expresses
approximately 6.times. more Idol mRNA (not shown). These mice can
be used to validate small molecule, shRNA, siRNA or antisense Idol
modulators.
[0380] We have also generated Idol knockout mice using the
targeting strategy shown in FIG. 17. Crosses of Idol-/+ mice have
yielded pups of all three expected genotypes at approximately
Mendelian ratios (FIG. 18). Thus, the global Idol null mice are
viable and available for use in screens of small molecule, shRNA,
siRNA, or antisense Idol modulators.
[0381] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
42150PRTHomo sapiens 1Lys Asn Trp Arg Leu Lys Asn Ile Asn Ser Ile
Asn Phe Asp Asn Pro 1 5 10 15 Val Tyr Gln Lys Thr Thr Glu Asp Glu
Val His Ile Cys His Asn Gln 20 25 30 Asp Gly Tyr Ser Tyr Pro Ser
Arg Gln Met Val Ser Leu Glu Asp Asp 35 40 45 Val Ala 50 250PRTPan
groglodytes 2Lys Asn Trp Arg Leu Lys Asn Ile Asn Ser Ile Asn Phe
Asp Asn Pro 1 5 10 15 Val Tyr Gln Lys Thr Thr Glu Asp Glu Val His
Ile Cys Arg Asn Gln 20 25 30 Asp Gly Tyr Ser Tyr Pro Ser Arg Gln
Met Val Ser Leu Glu Asp Asp 35 40 45 Val Ala 50 350PRTRattus
norvegicus 3Arg Asn Trp Arg Leu Arg Asn Ile Asn Ser Ile Asn Phe Asp
Asn Pro 1 5 10 15 Val Tyr Gln Lys Thr Thr Glu Asp Glu Ile His Ile
Cys Arg Ser Gln 20 25 30 Asp Gly Tyr Thr Tyr Pro Ser Arg Gln Met
Val Ser Leu Glu Asp Asp 35 40 45 Val Ala 50 450PRTMus musculus 4Arg
Asn Trp Arg Leu Lys Asn Ile Asn Ser Ile Asn Phe Asp Asn Pro 1 5 10
15 Val Tyr Gln Lys Thr Thr Glu Asp Glu Leu His Ile Cys Arg Ser Gln
20 25 30 Asp Gly Tyr Thr Tyr Pro Ser Arg Gln Met Val Ser Leu Glu
Asp Asp 35 40 45 Val Ala 50 550PRTOryctolagus cuniculus 5Lys Asn
Trp Arg Leu Arg Ser Val His Ser Ile Asn Phe Asp Asn Pro 1 5 10 15
Val Tyr Gln Lys Thr Thr Glu Asp Glu Val His Ile Cys Arg Ser Gln 20
25 30 Asp Gly Tyr Thr Tyr Pro Ser Arg Gln Met Val Ser Leu Glu Asp
Asp 35 40 45 Val Ala 50 632DNAMus musculus 6tccctactta acctacaatg
acctcaagtt tc 32732DNAMus musculus 7gaaacttgag gtcattgtag
gttaagtagg ga 32832DNAMus musculus 8tccctactta ttctacaatg
ttctcaagtt tc 32932DNAMus musculus 9gaaacttgag aacattgtag
aataagtagg ga 321051PRTHomo sapiens 10Leu Trp Lys Asn Trp Arg Leu
Lys Asn Ile Asn Ser Ile Asn Phe Asp 1 5 10 15 Asn Pro Val Gln Lys
Thr Thr Glu Asp Glu Val His Ile Cys His Asn 20 25 30 Gln Asp Gly
Tyr Ser Tyr Pro Ser Arg Gln Met Val Ser Leu Glu Asp 35 40 45 Asp
Val Ala 50 1156PRTHomo sapiens 11Met Trp Arg Asn Trp Gln His Lys
Asn Met Lys Ser Met Asn Phe Asp 1 5 10 15 Asn Pro Val Tyr Leu Lys
Thr Thr Glu Glu Asp Leu Ser Ile Asp Ile 20 25 30 Gly Arg His Ser
Ala Ser Val Gly His Thr Tyr Pro Ala Ile Ser Val 35 40 45 Val Ser
Thr Asp Asp Asp Leu Ala 50 55 1258PRTHomo sapiens 12Ile Trp Arg Asn
Trp Lys Arg Lys Asn Thr Lys Ser Met Asn Phe Asp 1 5 10 15 Asn Pro
Val Tyr Arg Lys Thr Thr Glu Glu Glu Asp Glu Asp Glu Leu 20 25 30
His Ile Gly Arg Thr Ala Gln Ile Gly His Val Tyr Pro Ala Arg Val 35
40 45 Ala Leu Ser Leu Glu Asp Asp Gly Leu Pro 50 55 1357PRTHomo
sapiens 13Asn Ser Asp Leu Lys Gly Pro Leu Thr Ala Gly Pro Thr Asn
Tyr Ser 1 5 10 15 Asn Pro Val Tyr Ala Lys Leu Tyr Met Asp Gly Gln
Asn Cys Arg Asn 20 25 30 Ser Leu Gly Ser Val Asp Glu Arg Lys Glu
Leu Leu Pro Lys Lys Ile 35 40 45 Glu Ile Gly Ile Arg Glu Thr Val
Ala 50 55 1457PRTHomo sapiens 14Leu Asp Ala Asp Phe Ala Leu Asp Pro
Asp Lys Pro Thr Asn Phe Thr 1 5 10 15 Asn Pro Val Tyr Ala Thr Leu
Tyr Met Gly Gly His Gly Ser Arg His 20 25 30 Ser Leu Ala Ser Thr
Asp Glu Lys Arg Glu Leu Leu Gly Arg Gly Pro 35 40 45 Glu Asp Glu
Ile Gly Asp Pro Leu Ala 50 55 1556PRTHomo sapiens 15Glu Tyr Ile Asn
Gln Ser Val Pro Lys Arg Pro Ala Gly Ser Val Gln 1 5 10 15 Asn Pro
Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser Arg Asp 20 25 30
Pro His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro Glu Tyr 35
40 45 Leu Asn Thr Val Gln Pro Thr Cys 50 55 1623DNAArtificialsiRNA
sequence 16aannnnnnnn nnnnnnnnnn ntt 231725DNAArtificialsiRNA
sequence 17aannnnnnnn nnnnnnnnnn nnntt 251823RNAArtificialsiRNA
sequence 18nannnnnnnn nnnnnnnnnn nnn 231923RNAArtificialsiRNA
sequence 19narnnnnnnn nnnnnnnnnn ynn 232028DNAArtificialPCR primer
20gctagcccta cttaacctac aatgacct 282128DNAArtificialPCR primer
21gctagcttct gatgcttcta cctctatc 282225DNAArtificialPCR primer
22gagctcagtt cccgggagct acacg 252350DNAArtificialPCR primer
23caccggagca aaggtgagag cttatcgaaa taagctctca cctttgctcc
502450DNAArtificialPCR primer 24aaaaggagca aaggtgagag cttatttcga
taagctctca cctttgctcc 502550DNAArtificialPCR primer 25caccgcctta
aactgagggt caagtcgaaa cttgaccctc agtttaaggc 502650DNAArtificialPCR
primer 26aaaagcctta aactgagggt caagtttcga cttgaccctc agtttaaggc
502750DNAArtificialPCR primer 27caccggacag cgaaggacag aaactcgaaa
gtttctgtcc ttcgctgtcc 502850DNAArtificialPCR primer 28aaaaggacag
cgaaggacag aaactttcga gtttctgtcc ttcgctgtcc 502950DNAArtificialPCR
primer 29caccgcatcg tgctcctgtt taagacgaat cttaaacagg agcacgatgc
503050DNAArtificialPCR primer 30aaaagcatcg tgctcctgtt taagattcgt
cttaaacagg agcacgatgc 503118DNAArtificialPCR primer 31cgaggactgc
ctcaacca 183224DNAArtificialPCR primer 32tgcagtccaa aatagtcaac ttct
243320DNAArtificialPCR primer 33ttcttcgtgg agcctcatct
203421DNAArtificialPCR primer 34cctccttgat gtgcaagaaa a
213520DNAArtificialPCR primer 35atgaggagct ctgtgccaag
203622DNAArtificialPCR primer 36tccttatgtt ttgcaacaat gc
223721DNAArtificialPCR primer 37tgtggagcct catctcatct t
213823DNAArtificialPCR primer 38agggactctt taatgtgcaa gaa
233922DNAArtificialPCR primer 39cagctatgag gacctgtgtg ag
224020DNAArtificialPCR primer 40tccttatgct tcgcaacgat
204122DNAArtificialPCR primer 41aggagatcaa ctccaccttc tg
224218DNAArtificialPCR primer 42atctgcagac cggacagg 18
* * * * *