U.S. patent application number 14/336457 was filed with the patent office on 2015-01-15 for farnesyltransferase inhibitors for treatment of laminopathies, cellular aging and atherosclerosis.
The applicant listed for this patent is The Government of the United States of America as represented by the Secretary of the Department of, Progeria Research Foundation, Inc., The Regents of the University of Michigan, The Government of the United States of America as represented by the Secretary of the Department of, The University of North Carolina at Chapel Hill. Invention is credited to Brian C. CAPELL, Francis S. COLLINS, Adrienne D. COX, Channing J. DER, Thomas GLOVER, Michael W. GLYNN, Leslie B. GORDON.
Application Number | 20150018381 14/336457 |
Document ID | / |
Family ID | 46329047 |
Filed Date | 2015-01-15 |
United States Patent
Application |
20150018381 |
Kind Code |
A1 |
GORDON; Leslie B. ; et
al. |
January 15, 2015 |
FARNESYLTRANSFERASE INHIBITORS FOR TREATMENT OF LAMINOPATHIES,
CELLULAR AGING AND ATHEROSCLEROSIS
Abstract
Although it can be farnesylated, the mutant lamin A protein
expressed in Hutchinson Gilford Progeria Syndrome (HGPS) cannot be
defarnesylated because the characteristic mutation causes deletion
of a cleavage site necessary for binding the protease ZMPSTE24 and
effecting defarnesylation. The result is an aberrant farnesylated
protein (called "progerin") that alters normal lamin A function as
a dominant negative, as well as assuming its own aberrant function
through its association with the nuclear membrane. The retention of
farnesylation, and potentially other abnormal properties of
progerin and other abnormal lamin gene protein products, produces
disease. Farnesyltransferase inhibitors (FTIs) (both direct
effectors and indirect inhibitors) will inhibit the formation of
progerin, cause a decrease in lamin A protein, and/or an increase
prelamin A protein. Decreasing the amount of aberrant protein
improves cellular effects caused by and progerin expression.
Similarly, treatment with FTIs should improve disease status in
progeria and other laminopathies. In addition, elements of
atherosclerosis and aging in non-laminopathy individuals will
improve after treatment with farnesyltransferase inhibitors.
Inventors: |
GORDON; Leslie B.; (Foxboro,
MA) ; COLLINS; Francis S.; (Rockville, MD) ;
GLOVER; Thomas; (Ypsilanti, MI) ; GLYNN; Michael
W.; (Darien, CT) ; CAPELL; Brian C.;
(Philadelphia, PA) ; COX; Adrienne D.; (Chapel
Hill, NC) ; DER; Channing J.; (Chapel Hill,
NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Government of the United States of America as represented by
the Secretary of the Department of
Progeria Research Foundation, Inc.
The Regents of the University of Michigan
The University of North Carolina at Chapel Hill |
Rockville
Peabody
Ann Arbor
Chapel Hill |
MD
MA
MI
NC |
US
US
US
US |
|
|
Family ID: |
46329047 |
Appl. No.: |
14/336457 |
Filed: |
July 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13857052 |
Apr 4, 2013 |
8828356 |
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14336457 |
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13567432 |
Aug 6, 2012 |
8691501 |
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13857052 |
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12905838 |
Oct 15, 2010 |
8257915 |
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13567432 |
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11828117 |
Jul 25, 2007 |
7838531 |
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12905838 |
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PCT/US2006/002977 |
Jan 27, 2006 |
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11828117 |
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10943400 |
Sep 17, 2004 |
7297492 |
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11828117 |
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PCT/US03/33058 |
Oct 17, 2003 |
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10943400 |
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60648307 |
Jan 28, 2005 |
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60707192 |
Aug 9, 2005 |
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60463084 |
Apr 14, 2003 |
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60419541 |
Oct 18, 2002 |
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Current U.S.
Class: |
514/290 ;
514/303; 514/312 |
Current CPC
Class: |
C12Q 2600/136 20130101;
A61P 43/00 20180101; C12Q 1/6886 20130101; C12Q 2600/156 20130101;
G01N 2500/04 20130101; G01N 2333/91171 20130101; G01N 2500/10
20130101; C12Q 1/6883 20130101; G01N 33/68 20130101; A61K 45/06
20130101; A61K 31/4545 20130101; C07K 14/47 20130101; A61K 31/4709
20130101 |
Class at
Publication: |
514/290 ;
514/303; 514/312 |
International
Class: |
A61K 31/4545 20060101
A61K031/4545; A61K 31/4709 20060101 A61K031/4709 |
Claims
1. A method of reducing at least one clinical symptom of
Hutchinson-Guilford Progeria Syndrome (HGPS) in a subject having
HGPS, the method comprising administering to the subject a
therapeutically effective dose of a farnesyltransferase inhibitor
(FTI).
2. The method of claim 1, wherein the clinical symptom of HGPS
comprises one or more of failure to thrive, maldevelopment,
cardiovascular disease, abnormal bone density, distal bone
resorption, osteoporosis, or decreased adipose tissue.
3. The method of claim 2, wherein the clinical symptom is a
cardiovascular disease comprising one or more of atherosclerosis,
atherosclerotic plaques in large or small arteries, interstitial
fibrosis, stenosis, or paucity of medial smooth muscle cells.
4. The method of claim 1, wherein the method prevents or retards
progression of HGPS in the subject.
5. The method of claim 1, wherein the FTI is PD169541, R115777,
SCH66336, L-744832, FTI-2153 or a derivative thereof that maintains
farnesyltransferase inhibitory activity.
6. The method of claim 1, further comprising administering a second
therapeutic compound to the subject.
7. The method of claim 6, wherein the second therapeutic compound
is not a farnesyltransferase inhibitor (FTI).
8. The method of claim 1, wherein the therapeutically effective
amount of the FTI is about 0.1 nM per kilogram (kg) body weight to
about 1 .mu.M per kg body weight, such as about 1 nM to about 500
nM per kg body weight, or about 5 nM to about 50 nM per kg body
weight.
9. The method of claim 1, wherein the therapeutically effective
amount of the FTI is 0.1 to 200 mg/kg body weight.
10. The method of claim 9, wherein the therapeutically effective
amount of the FTI is 1.0, 10, 25, 50, or 100 mg/kg body weight.
11. The method of claim 1, wherein the FTI is lonafarnib
(SCH66336).
12. The method of claim 11, wherein the therapeutically effective
amount of SCH66336 is at least 115 mg/m.sup.2 BID.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of co-pending U.S. patent application
Ser. No. 13/857,052, filed Apr. 4, 2013; which is a continuation of
U.S. patent application Ser. No. 13/567,432, filed Aug. 6, 2012,
which issued as U.S. Pat. No. 8,691,501 on Apr. 8, 2014; which is a
division of U.S. patent application Ser. No. 12/905,838, filed Oct.
15, 2010, which issued as U.S. Pat. No. 8,257,915 on Sep. 4, 2012;
which is a division of U.S. patent application Ser. No. 11/828,117,
filed Jul. 25, 2007, which issued as U.S. Pat. No. 7,838,531 on
Nov. 23, 2010; which is a continuation-in-part of International
Application No. PCT/US2006/002977, filed Jan. 27, 2006; which in
turn claims the benefit of U.S. Provisional Application No.
60/648,307, filed Jan. 28, 2005 and U.S. Provisional Application
No. 60/707,192, filed Aug. 9, 2005; and a continuation-in-part of
U.S. Utility application Ser. No. 10/943,400, filed Sep. 17, 2004,
which issued as U.S. Pat. No. 7,297,942 on Nov. 20, 2007; which is
a continuation of International Application No. PCT/US2003/033058,
filed Oct. 17, 2003, which in turn claims the benefit of U.S.
Provisional Application No. 60/463,084, filed Apr. 14, 2003, and
U.S. Provisional Application No. 60/419,541, filed Oct. 18, 2002.
Each of these applications is incorporated herein in their
entirety.
FIELD
[0002] This disclosure relates to treatment of laminopathies,
cellular aging and aging-related conditions, and more particularly
to the use of farnesyltransferase inhibitors (FTIs) and other
compounds and compositions to treat such conditions. It also
relates to methods of identifying agents useful in treating, for
instance, laminopathies.
BACKGROUND
[0003] Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare
genetic disease that affects children in the first decade of life
and causes a remarkable phenotype resembling many aspects of aging.
Affected children develop an extremely aged appearance, a lack of
subcutaneous fat, growth retardation and severe atherosclerosis.
Affected children die of premature atherosclerosis at an average
age of 13 years. Progeria is a disease in which some, but not all,
of its manifestations (in vivo and in vitro) represent a model of
accelerated aging (reviewed in Sweeney & Weiss, Gerontology
38:139-152, 1992). Clinical features common to progeria and normal
aging include alopecia (although the pattern of hair loss differs),
sclerodermatosis, atherosclerosis, lipofuscin deposition, nail
dystrophy, hypermelanosis, decreased adipose tissue, and
osteoporosis. Clinical differences include sequelae of
maldevelopment in progeria, with coxa valga, distal bone
resorption, delayed dentition, facial disproportion, failure to
thrive, and short stature. Features of aging that are absent in
progeria include neurosensory decline such as Alzheimer's disease,
dementia, hearing loss, and presbyopia.
[0004] Recently, the gene responsible for HGPS was identified, and
HGPS joined a group of syndromes--the laminopathies--all of which
have an underlying defect in the lamin A/C gene (LMNA) (Eriksson et
al., Nature 423:293-298, 2003). LMNA codes for the lamin A and
lamin C isoforms, which differ due to alternate splicing, as well
as the .DELTA.10 isoform found in sperm. The lamins are a component
of the nuclear lamina, a fibrous matrix located at the interior of
the nuclear membrane, responsible for nuclear integrity and
organization. In addition, lamins are also present in the
nucleoplasm and may be involved in more complex spatial
organization of the nucleus. They play a role in a wide array of
nuclear processes, including transcription, replication, chromatin
organization, nuclear shape, cell division, and cell cycle
functions (Gruenbaum et al., J Struct Biol 129:313-23, 2000;
Gruenbaum et al., Nat. Rev. Mol. Cell Biol. 6:21-31, 2005). The
LMNA gene is primarily expressed in differentiated tissues in the
fetus and adult and may be important in maintaining the
differentiated state (Rober et al., Development 105:365-378, 1989).
In fact, lamin A expression is down-regulated in many tumors,
perhaps as part of the loss of differentiation seen in those tumors
(Muller et al., Leukemia 8:940-945, 1994).
[0005] The pre-lamin A protein contains a CAAX box (SEQ ID NO: 31)
at the carboxy terminus, which is an invariant cysteine followed by
two aliphatic amino acids with the X denoting the terminal amino
acid. The CAAX box signals for isoprenylation, the addition of a
15-carbon farnesyl isoprenoid lipid group to the cysteine by the
enzyme farnesyltransferase (FTase) or a 20-carbon geranylgeranyl
isoprenoid lipid by geranylgeranyltransferase I (GGTaseI) (Beck et
al., J. Cell Biol. 110:1489-1499, 1990). The final amino acid
defines the specificity for the addition of the isoprenyl group
with methionine, serine, glutamine, or alanine signaling
farnesylation and leucine signaling the addition of a 20-carbon
geranylgeranyl isoprenoid group catalyzed by the structurally
related enzyme GGTase I (Moores et al., J Biol Chem
266:14603-14610, 1991; Cox & Der, Curr. Opin. Pharmacol.
2:388-393, 2002). The native lamin A CAAX box (SEQ ID NO: 31)
consists of CSIM (cysteine, serine, isoleucine, methionine) (SEQ ID
NO: 32). Farnesylation, together with subsequent CAAX-signaled
modifications, promote prelamin A association with the nuclear
membrane (Hennekes & Nigg, J. Cell Sci. 107:1019-1029, 1994).
Farnesylation is a permanent modification; once a farnesyl group is
added to a protein, it remains attached to that residue for the
life of the protein. Following farnesylation, the terminal three
AAX amino acids are removed, and the C-terminal isoprenylated
cysteine undergoes methyl esterification (Hennekes & Nigg, J.
Cell Sci. 107:1019-1029, 1994). While both B-type lamins and lamin
A are farnesylated and carboxymethylated, unique to lamin A is a
second cleavage that occurs inside the nucleus causing the removal
of an additional 15 C-terminal amino acids from the mature protein,
including the farnesylated cysteine. Because farnesylation is a
permanent posttranslational modification, proteolytic cleavage of
the farnesylated cysteine is necessary for full processing of the
prelamin A protein to mature lamin A, and for its correct
subcellular localization and function. Thus, this final cleavage
step and the resulting loss of the farnesyl anchor presumably
releases prelamin A from the nuclear membrane and allows it to be
inserted into the nuclear lamina. In HGPS, although preprogerin can
be farnesylated, its internal deletion of amino acids 606-656
removes the endoprotease recognition site necessary for executing
the final cleavage step. This final cleavage step appears to be
important for normal function as mutations in ZMPSTE24 cause a
severe form of mandibuloacral dysplasia (MADB), one of the
laminopathies which is phenotypically similar to HGPS (Agarwal et
al., Hum. Mol. Genet. 12:1995-2001, 2003). ZMPSTE24 is the human
homolog of yeast STE 24 and is responsible for the final cleavage
of lamin A that removes the 15 terminal amino acids (Pendas et al.,
Nat. Genet. 31:94-99, 2002).
[0006] Nearly all HGPS patients have the same silent mutation
(G608G) creating an abnormal splice donor site in exon 11 of the
LMNA gene (Eriksson et al., Nature 423:293-298, 2003), which causes
a 150 base pair mRNA deletion in the lamin A transcript. The result
of the mis-splicing is a protein missing 50 amino acids near the
C-terminus (henceforth called "preprogerin" prior to
posttranslational processing and "progerin" after
post-translational processing). The deleted region includes the
protein cleavage site that normally removes the C-terminal 15 amino
acids, including the farnesylated cysteine. The deleted region also
contains two potential cyclin-dependent kinase target serines (652
and 657) that may be involved in dissociation and reassociation of
the nuclear membrane at each cell division (Sinensky et al., J Cell
Sci 107(Pt 1):61-7, 1994; Kilic et al., J Biol Chem
272(8):5298-304, 1997) and it may affect molecular solubility
(Hennekes & Nigg, J Cell Sci. 107:1019-29, 1994).
SUMMARY OF THE DISCLOSURE
[0007] Although it can be farnesylated, the progerin protein cannot
be defarnesylated because the characteristic mutation causes
deletion of the second cleavage site necessary for binding ZMPSTE24
and effecting defarnesylation (Eriksson et al., Nature 423:293-298,
2003). The result is a shortened, aberrant (abnormally)
farnesylated protein capable of altering normal lamin A function as
a dominant negative, as well as assuming its own aberrant function
through its association with the nuclear membrane. Thus, the
multisystem disease process and the variety of genes that are
affected in HGPS (Csoka et al., Aging Cell 3:235-243, 2004) lie
downstream of a protein defect that is central to basic cellular
function. Other lamin defects also lead to disease processes
(generally referred to as laminopathies) due to aberrant properties
of the LMNA protein product.
[0008] The retention of farnesylation, and potentially other
abnormal properties of progerin and other abnormal lamin gene
protein products, produces disease. Farnesyltransferase inhibitors
(both direct effectors and indirect inhibitors of
farnesyltransferase) will prevent farnesylation, inhibit the
formation of progerin, cause a decrease in lamin A protein and an
increase in prelamin A and preprogerin proteins. Decreasing the
amount of aberrant protein will improve disease status in Progeria
and other laminopathies (in that the status will be moved towards
normal). In addition, altering lamin A using farnesyltransferase
inhibitors (both direct effectors and indirect inhibitors) will
improve those elements of other diseases and conditions, such as
atherosclerosis and aging, in normal individuals that involve lamin
A.
[0009] The foregoing and other features and advantages will become
more apparent from the following detailed description of several
embodiments, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1 shows a schematic overview of normal lamin A
processing. The C-terminal CAAX box (SEQ ID NO: 31) of prelamin A
undergoes farnesylation, which allows localization of the partially
processed protein to the nuclear membrane. The AAX endopeptidase
cleaves the last three amino acids from the farnesylated protein.
Prelamin A then undergoes methyl esterification at the C-terminus.
The endoprotease ZMPSTE24 then cleaves the c-terminal 15 amino
acids, which cleavage releases a short peptide containing both the
farnesylation and the methyl ester. This last proteolytic cleavage
is prevented in progerin, as the cleavage site is lost in the 50
amino acid deletion. In addition, neither proteolytic cleavage can
occur without prior farnesylation of the precursor protein.
[0011] FIG. 2 is a diagram showing the differential processing of
lamin A exon 11 in wild-type and the HGPS mutant. The silent
mutation G608G leads to activation of a cryptic splice site, which
results in splicing-out of 150 nucleotides corresponding to the
3'-end of Exon 11.
[0012] FIG. 3 shows drawings of prelamin A, lamin A, and progerin.
The deletion in progerin removes amino acids 608-657 of the
wildtype lamin A protein; this region includes both a
phosphorylation site (indicated in the figure by an asterisk) and a
proteolytic cleavage site that is required for maturation from the
pre-form to the mature lamin A. The nuclear localization sequence
(NLS) is unaffected by the mutation.
[0013] FIG. 4A-C illustrates nuclear morphology and protein
localization in cells transfected with plasmid DNA encoding green
fluorescent protein (GFP)-tagged lamin A or GFP-tagged wild type or
mutant progerin proteins. GFP-lamin A (panels 4A and 4B) or
GFP-progerin (panel 4C) expression constructs were transiently
transfected into the normal fibroblast line, GM08398, using
FUGENE.RTM. transfection reagent (Roche). Seventy-two hours later
the cells were fixed with 4% paraformaldehyde and visualized using
a Zeiss AXIOPHOT.TM. fluorescence microscope with a SENSYS.RTM. CCD
camera and Applied Imaging digital imaging capture software. The
actin cytoskeleton was stained with Rhodamine-phalloidin. Panel 4A
shows the same cell as in Panel B but with the focal plane
bisecting the nucleus. In 4B and 4C the periphery of the nucleus is
in focus to show the altered localization of GFP-progerin in panel
4C.
[0014] FIG. 5A-D illustrates immunocytochemistry using a lamin
A-specific antibody to detect localization of endogenous lamin A.
Normal (5A and 5B) and HGPS (5C and 5D) fibroblasts were grown on
coverslips, fixed with formaldehyde and probed with anti-lamin A
antibodies (USBiological). Lamin A aggregates were observed in HGPS
fibroblasts. Images show two focal planes, a nuclear cross-section
(5A and 5C) and a focal plane bisecting the nucleus (5B and
5D).
[0015] FIG. 6 A-B) shows images of confocal microscopy of
transfected cells. Normal fibroblasts were plated on coverslips,
transfected, and fixed as in FIG. 4. Localization of GFP-lamin A
(panel 6A) and progerin (panel 6B) was analyzed using a Zeiss LSM
510 confocal microscope mounted on a Zeiss AXIOVERT.RTM. 100M
inverted microscope. Three focal planes are shown--top, middle and
bottom (from left to right). These images show the location of the
GFP-progerin aggregates as thicker regions at the nuclear periphery
(6B). Fibroblasts expressing normal GFP-lamin A have GFP signal
around the nuclear periphery (6A).
[0016] FIG. 7A-E illustrates the effect of a 72 hour exposure to
100 nM FTI (PD169451) on GFP-lamin A (7A) and GFP-progerin (7B)
localization, protein processing (7C) and nuclear morphology (7D
and 7E). Confocal microscopy was used to examine the effects of
FTIs on transiently-expressed GFP-lamin A (7A) and GFP-progerin
(7B) in normal fibroblasts. Three focal planes are shown--top,
middle and bottom (from left to right). Exposure to 100 nM FTI for
72 hours caused about half of the endogenous lamin A to be
unprocessed pre-lamin A in normal and HGPS fibroblasts (7C).
Exposure to the FTI increased the percent of mock transfected cells
and GFP-lamin A expressing cells with abnormal nuclear morphology
(7D). However, the percentage of cells expressing GFP-progerin with
an abnormal nuclear morphology decreased by 33% when exposed to the
FTI for 72 hours. The nuclear morphology of HGPS fibroblasts showed
a similar improvement in the percent of abnormal nuclei when
exposed to the FTI for 72 hours (7E).
[0017] FIG. 8A-C shows the nuclear morphology of normal fibroblasts
(GM08398) expressing GFP-lamin A (panel 8A), GFP-progerin (panel
8B), and GFP-lamin A L647R (panel 8C). DAPI staining of nuclei is
shown on the left and GFP signal on the right. The GFP constructs
were expressed from the pRevTRE retroviral TET-OFF.RTM. expression
vector for three days by removing tetracycline from the growth
medium followed by fixing the cells with 4% paraformaldehyde and
mounting with PROLONG.RTM. Gold antifade mounting reagent with
DAPI.
[0018] FIG. 9 illustrates the effect of expressing the cleavage
minus mutant (GFP-lamin A L647R) on nuclear morphology. GFP-lamin
A, GFP-progerin, and GFP-lamin A L647R were expressed in the normal
fibroblast line, GM08398, for three days followed by fixing the
cells with 4% paraformaldehyde and mounting with ProLong Gold with
DAPI. Abnormal nuclear morphology was assessed as nuclei with
multiple blebs. The elevated proportion of abnormal nuclei in the
presence of tetracycline (+Tet) in the GFP-progerin and GFP-LA
L647R is due to the leaky expression of these proteins in the
concentration of tetracycline used to prevent expression.
[0019] FIG. 10 illustrates the effect of long-term exposure to low
doses of FTI (PD169451) on the nuclear morphology of normal
(GM08398 and HGMDFN090) and HGPS (AG11498, P01, HGADFN136 and
HGADFN004) fibroblasts. Fibroblasts were maintained in medium with
0 nM, 10 nM or 100 nM FTI. Cultures were plated in chambered
microscope slides 24 hours prior to fixing with 4% paraformaldehyde
for nuclear morphology studies. **--Too few cells to count. The
HGADFN004 line failed to grow in 100 nM FTI to the 14 day time
point. All cell lines were passage 6 at the beginning of the
analysis except for HGADFN004, which was at passage 12.
[0020] FIG. 11A-D illustrates the effect of the FTI, PD169451, on
the nuclear morphology of normal fibroblasts (GM08398) stably
expressing GFP-lamin A, GFP-progerin and GFP-LA(L647R) using a
retroviral TET-OFF.RTM. expression vector system (Clontech).
Fibroblasts were maintained in medium with 0 nM, 10 nM or 100 nM
FTI for 6 days.
[0021] Cultures were plated in chambered microscope slides 24 hours
prior to fixing with 4% paraformaldehyde for nuclear morphology
studies. Cells were maintained in three predetermined tetracycline
concentrations, 0 mM for full expression (11A), 0.05 mM for
intermediate expression levels (11B), and 8 mM for very low
expression levels (11C). Using a TET-OFF.RTM. expression vector
system allowed for modulation of expression levels with
tetracycline concentration as demonstrated by immunoblot of cells
from the above experiment (11D). When GFP-progerin and
GFP-LA(L647R) mutant was stably expressed in normal fibroblasts at
intermediate levels (0.05 mg/ml tetracycline), 10 nM FTI
substantially reduced the percent abnormal nuclei by 71% and 67%,
respectively (p<0.0001) (11B). Analysis of nuclear morphology
was performed in a double blinded fashion with at least 70 cells
analyzed per sample.
[0022] FIG. 12A-I illustrates the effect of FTI on the distribution
of GFP signal in normal fibroblasts (GM08398) expressing GFP-lamin
A (panels 12A, 12B and 12C), GFP-progerin (12D, 12E and 12F) and
GFP-LA(L647R) (12G, 12H, and 12I) using a retroviral TET-OFF.RTM.
expression vector system with various FTI concentrations.
Fibroblasts were maintained in medium with 0 nM (12A, 12D, 12G), 10
nM (12B, 12E, and 12H) or 100 nM (12C, 12F and 12I) FTI for six
days. Expression levels of the GFP fusions were maintained at
levels similar to endogenous levels of lamin A by the addition of
0.05 mg/ml tetracycline. Signals were visualized with a Zeiss
AXIOPHOT.TM. Fluorescent microscope and imaging was performed using
the Olympus DP70 Digital Camera System.
[0023] FIG. 13 includes the maps of three constructs discussed in
Examples 5 and 6. MAP 1 illustrates a representative cDNA construct
expressing EGFP-labeled lamin A, with or without the 150-nucleotide
deletion used to mimic HGPS lamin A. The construct is expressed
under the control of a CMV promoter, and also contains a myc
epitope tag. MAP 2 illustrates a representative minigene construct
expressing EGFP-labeled lamin A, with or without the 150-nucleotide
deletion used to mimic HGPS lamin A. The construct is expressed
under the control of a CMV promoter, and also contains a myc
epitope tag. MAP 3 illustrates a representative tet-inducible
minigene construct expressing lamin A, with or without the
150-nucleotide deletion used to mimic HGPS lamin A (Tet 75/77-lamin
A minigene constructs). One specific example of a Tet-inducible
minigene construct is Tet 75-lamin A, which contains the G608G
mutation (GCG to GGT) causing partial aberrant splicing from Exon
11 to Exon 12 and deleting 150 nucleotides (corresponding to 50
amino acid residues), thereby producing a progerin-like protein.
Another is Tet 77-lamin A, which contains the wild type lamin A
minigene.
[0024] FIG. 14 is a series of schematic drawings of lamin A and
mutant lamin A expression constructs.
[0025] FIG. 15 is a Western blot illustrating expression of
Progerin from fibroblasts transfected with Progerin cDNA and
minigene: M=Molecular weight marker; C1 Normal Fibroblast
untransfected; GFP-P=Normal Fibroblast transfected with complete
Progerin cDNA construct; GFP-MG6=Normal Fibroblast transfected with
MG6-minigene which shows the splicing event.
[0026] FIG. 16 A-C) is a series of digital scans of nucleic acid
gels, showing processing of lamin and progerin minigene constructs
in human vascular cells. FIG. 16A shows results in EC; FIG. 16B
shows coronary artery SMC; and FIG. 16C shows radial artery SMC.
Each cell type expresses and processes progerin after minigene
transcript transfection. Cells were electroporated and exposed to
either Progerin minigene (MG) or LMNA-lamin A (L). Arrows indicate
the 489 base pair band of interest in the MG lane, produced by the
Progeria gene which indicates a splicing event occurring in VSMC
and EC. Other bands indicate incomplete splicing of the
minigene.
[0027] FIG. 17 shows population doubling times of cultured human
dermal fibroblast lines. Solid lines indicate 4-parameter
exponential growth curve fit for HGPS (line through triangles) and
Normal (line through circles). Passage doubling (PD) time at each
passage is indicated by open triangles (HGPS) and open circles
(Normal). Filled diamond indicates a second HGPS cell line at later
passage for comparison. Corresponding SA.beta.-gal staining is
indicated.
[0028] FIG. 18 A-C Cells were stained with Annexin V for apoptosis,
Propidium Iodide (PI) for necrosis, and DAPI for DNA (all
20.times.). FIG. 18A: Non-apoptotic normal fibroblasts (passage 14)
display round nuclei. FIG. 18B: HGPS fibroblasts (passage 14)
display apoptosis with underlying nuclei. FIG. 18C: HGPS
fibroblasts immortalized with hTERT catalytic subunit of telomerase
show no apoptosis (passage 35).
[0029] FIG. 19A-D Cells were plated on glass coverslips and stained
for lamin A/C (upper and lower panels) and DAPI (lower panels).
Images were taken under 40.times. objective and analyzed for
nuclear morphology. HGPS cells (19B & 19D) exhibit blebbing of
the nuclear envelope whereas Normal cells (19A & 19C) do
not.
[0030] FIG. 20 Normal (n=6) and HGPS (n=6) fibroblast cell lines
were assessed for MMP-3 secretion. MMP-3 secretion from HGPS
fibroblasts was significantly (p=0.003) decreased over
controls.
[0031] FIG. 21 Translation of the LMNA gene yields the prelamin A
protein, which requires posttranslational processing for
incorporation into the nuclear lamina. The prelamin protein
contains a CAAX box (CSIM) (SEQ ID NO: 31) at the C-terminus
signaling isoprenylation, in this case addition of a farnesyl group
to the cysteine by the enzyme farnesyltransferase. Following
farnesylation, the terminal three amino acids (SIM) are cleaved,
and the terminal farnesylated cysteine undergoes methyl
esterification. A second cleavage step by the ZMPSTE24 endoprotease
then removes the terminal 15 amino acids, including the farnesyl
group. This final cleavage step is blocked in progeria.
[0032] FIG. 22A-F Subcellular localization and induction of nuclear
blebbing by prenylation mutants of wild-type and progerin lamin A.
Transient transfection of HeLa cells with expression vectors
encoding the indicated wild-type or progerin lamin A protein. (22A)
Normal wildtype lamin A-CSIM CAAX motif (SEQ ID NO: 31); (22B)
wild-type lamin A with the CSIM sequence (SEQ ID NO: 32) mutated to
SSIM (SEQ ID NO: 33) to prevent farnesylation (because serine (S)
cannot be farnesylated), relocalizing all lamin A into
nucleoplasmic aggregates; (22C) wild-type lamin A with the CSIM
sequence (SEQ ID NO: 32) mutated to CSIL (SEQ ID NO: 34), predicted
to be geranylgeranylated by GGTase I, resulting in a mix of
nucleoplasmic and nuclear membrane localization; (22D) the
progerin-CSIM construct recreates the hallmark nuclear blebbing
seen in HGPS; (22E) when the CSIM (SEQ ID NO: 32) of the
progerin-CSIM is mutated to SSIM (SEQ ID NO: 33) (progerin-SSIM),
blebbing is prevented, and progerin accumulates in nucleoplasmic
aggregates; (22F) progerin-CSIL causes blebbing indistinguishable
from that of authentic progerin-CSIM. Cells were visualized 48
hours after transfection and photomicrographs were taken at
60.times. magnification.
[0033] FIG. 23A-F FTI treatment prevents nuclear blebbing caused by
farnesylated, but not geranylgeranylated, progerin. HeLa cells were
transiently transfected with expression vectors encoding either the
progerin-CSIM, progerin-SSIM or progerin-CSIL constructs, and were
treated with either 0 or 2 .mu.M of the FTI lonafarnib/SCH66336.
(23A) The progerin-CSIM construct recreates the hallmark nuclear
blebbing seen in HGPS, while FTI treatment almost totally abolishes
it, and leads to relocalization of progerin into nucleoplasmic
aggregates (23D); (23B) unprocessed progerin-SSIM mimics the
phenotype of FTI-treated cells expressing authentic progerin-CSIM,
with all progerin relocalized into nucleoplasmic aggregates, and
this does not change significantly with treatment (23E); (23C)
progerin-CSIL, predicted to be geranylgeranylated, recreates
nuclear blebbing but is resistant to FTI treatment (23F). Cells
were visualized 48 hours after transfection and photomicrographs
were taken at 60.times. magnification.
[0034] FIG. 24: The percentage of blebbed HeLa nuclei when
transiently transfected with the above four constructs and treated
with a single dose of lonafarnib/SCH66336 at the time of
transfection. Percentages were read by three independent observers,
who scored 200 cells for each experiment and were blind to the
construct being used. Error bars show the standard error of the
mean, and the p-values are for the comparison of the treated cells
to the untreated control. Wild-type lamin A-CSIM does not change
significantly from the typical 5% nuclear blebbing percentage of
normal cells. The progerin-CSIM shows a dramatic dose-response. The
progerin-CSIL, expected to be geranylgeranylated, is resistant to
inhibition of farnesylation. The progerin-SSIM, which cannot be
farnesylated or geranylgeranylated, demonstrates virtually no
blebbing.
[0035] FIG. 25: FTI treatment causes reversion of the nuclear
blebbing in two different progerin-expressing HGPS human
fibroblasts. Cells were stained with anti-lamin A antibody after
being treated for 3 days with either 0, 1, or 2 .mu.M of the FTI
lonafarnib/SCH66336 (60.times.).
[0036] FIG. 26: The percentage of blebbed nuclei when HGPS skin
fibroblasts were treated once daily for three days with the above
doses of lonafarnib. AG06299 is from the 34 year-old unaffected
mother (passage 31) of AG06917, an affected 6 year-old (passage
14). AG11513 is from an affected 8 year-old (passage 7), and
AG11498 is from an affected 14 year-old (passage 11). Percentages
were read by three independent observers, who scored 200 cells for
each experiment and were blind to the genotype of the fibroblasts
and the dose of the FTI being used. Error bars show the standard
error of the mean, and the p-values are for the comparison of the
treated cells to the untreated control.
[0037] FIG. 27: FTI treatment alone is sufficient to impair
progerin processing. NIH-3T3 cells transiently expressing empty
vector ("v.o."), or GFP-tagged progerin-CSIM ("Progerin") or
GFP-tagged progerin-SSIM ("SSIM") were treated for 48 hours with
vehicle (DMSO) or inhibitors of FTase or GGTase I (5 .mu.M FTI-2153
or GGTI-2166, respectively). Total cell lysates were resolved on
SDS-PAGE and immunoblotted with anti-GFP to detect
GFP-progerin-CSIM or the fully unprocessed mutant
GFP-progerin-SSIM. Treatment with FTI, but not GGTI, induces a
mobility shift of progerin-CSIM to that of the unprocessed
progerin-SSIM, demonstrating that progerin is a FTI target that
does not undergo alternative prenylation and modification by GGTase
I. Schematic diagrams at the top of each lane indicate the
molecular entity that constitutes the major species in each
experiment. P=progerin sequence up to CAAX motif (SEQ ID NO: 31);
C=cysteine; F=farnesyl group.
SEQUENCE LISTING
[0038] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. The Sequence Listing is
submitted as an ASCII text file, created on Jul. 19, 2014,
.about.56 KB, which is incorporated by reference herein. Only one
strand of each nucleic acid sequence is shown, but the
complementary strand is understood as included by any reference to
the displayed strand. In the accompanying sequence listing:
[0039] SEQ ID NOs: 1 and 2 are primers (laminAF1 and laminAR1,
respectively) used to amplify normal and mutant lamin A/C.
[0040] SEQ ID NOs: 3-8 are primers used to introduce the L647R
mutant into the pRevTRE-GFP-lamin A construct.
[0041] SEQ ID NOs: 9 and 10 are a pair of forward and reverse
primers.
[0042] SEQ ID NOs: 11-18 are primers used to generate lamin A CAAX
mutants.
[0043] SEQ ID NO: 19 is the CSIM Human lamin A (LMNA) wt mRNA
coding sequence (nucleotide residues 213-2207 of GenBank Accession:
NM.sub.--170707).
[0044] SEQ ID NO: 20 is the CSIM LMNA protein translation.
[0045] SEQ ID NO: 21 is the CSIL Human lamin A (LMNA) wt mRNA
coding sequence (nucleotide residues 213-2207 of GenBank Accession:
NM.sub.--170707).
[0046] SEQ ID NO: 22 is the CSIL LMNA protein translation.
[0047] SEQ ID NO: 23 is the SSIM Human lamin A (LMNA) wt mRNA
coding sequence (nucleotide residues 213-2207 of GenBank Accession:
NM.sub.--170707).
[0048] SEQ ID NO: 24 is the SSIM LMNA protein translation.
[0049] SEQ ID NO: 25 is the CSIM Human lamin A (LMNA) del 150 mRNA
coding sequence.
[0050] SEQ ID NO: 26 is the CSIM LMNA_del50 protein
translation.
[0051] SEQ ID NO: 27 is the Human lamin A (LMNA) CSIL del 150 mRNA
coding sequence.
[0052] SEQ ID NO: 28 is the CSIL LMNA_del50 protein
translation.
[0053] SEQ ID NO: 29 is the SSIM Human lamin A (LMNA) del 150 mRNA
coding sequence.
[0054] SEQ ID NO: 30 is the SSIM LMNA_del50 protein
translation.
[0055] SEQ ID NOs: 31-34 are the CAAX box motif of the prelamin
protein and variants thereof.
DETAILED DESCRIPTION
I. Abbreviations
[0056] EC: endothelial cells
[0057] EGFP: enhanced green fluorescent protein
[0058] FPP: farnesyl pyrophosphate
[0059] FTase: farnesyltransferase
[0060] FTI: farnesyltransferase inhibitor
[0061] GFP: green fluorescent protein
[0062] GGTase I: geranylgeranyltransferase
[0063] GGTI: geranylgeranyltransferase inhibitor
[0064] HGPS: Hutchinson-Gilford Progeria Syndrome
[0065] ICC: immunocytochemistry
[0066] LA: lamin A
[0067] LMNA: gene encoding lamin A and lamin C
[0068] MADB: mandibuloacral dysplasia
[0069] MG: minigene
[0070] ORF: open reading frame
[0071] PD: Passage doubling
[0072] RT-PCR: reverse-transcription polymerase chain reaction
[0073] UTRs: untranslated regions
[0074] VSMC: vascular smooth muscle cells
II. Terms
[0075] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0076] In order to facilitate review of the various embodiments of
the invention, we provide the following information concerning some
of the terms used herein. Additional information may be found in
the literature, including the references cited and incorporated by
reference herein.
[0077] Abnormal: Deviation from normal characteristics. Normal
characteristics can be found in a control, a standard for a
population, etc. For instance, where the abnormal condition is a
disease condition, such as progeria, a few appropriate sources of
normal characteristics might include an individual who is not
suffering from the disease (e.g., progeria), a population standard
of individuals believed not to be suffering from the disease,
etc.
[0078] Likewise, abnormal may refer to a condition that is
associated with a disease. The term "associated with" includes an
increased risk of developing the disease as well as the disease
itself. For instance, a certain abnormality (such as an abnormality
in an LMNA nucleic acid or Lamin protein expression) can be
described as being associated with the biological conditions of
progeria and tendency to develop premature aging disease or
condition.
[0079] An abnormal nucleic acid, such as an abnormal LMNA nucleic
acid, is one that is different in some manner to a normal
(wildtype) nucleic acid. Such abnormality includes but is not
necessarily limited to: (1) a mutation in the nucleic acid (such as
a point mutation (e.g., a single nucleotide polymorphism) or short
deletion or duplication of a few to several nucleotides); (2) a
mutation in the control sequence(s) associated with that nucleic
acid such that replication or expression of the nucleic acid is
altered (such as the functional inactivation of a promoter); (3) a
decrease in the amount or copy number of the nucleic acid in a cell
or other biological sample (such as a deletion of the nucleic acid,
either through selective gene loss or by the loss of a larger
section of a chromosome or under expression of the mRNA); (4) an
increase in the amount or copy number of the nucleic acid in a cell
or sample (such as a genomic amplification of part or all of the
nucleic acid or the overexpression of an mRNA), each compared to a
control or standard; and (5) an alteration in a sequence that
controls the splicing mechanism, in such a way that either a normal
splice signal is inactivated or an abnormal splice signal is
created. It will be understood that these types of abnormalities
can co-exist in the same nucleic acid or in the same cell or
sample; for instance, a genomic-amplified nucleic acid sequence may
also contain one or more point mutations. In addition, it is
understood that an abnormality in a nucleic acid may be associated
with, and in fact may cause, an abnormality in expression of the
corresponding protein.
[0080] Abnormal protein expression, such as abnormal lamin A
protein expression, refers to expression of a protein that is in
some manner different to expression of the protein in a normal
(wildtype) situation. This includes but is not necessarily limited
to: (1) a mutation in the protein such that one or more of the
amino acid residues is different; (2) a short deletion or addition
of one or a few amino acid residues to the sequence of the protein;
(3) a longer deletion or addition of amino acid residues, such that
an entire protein domain or sub-domain is removed or added; (4)
expression of an increased amount of the protein, compared to a
control or standard amount; (5) expression of an decreased amount
of the protein, compared to a control or standard amount; (6)
alteration of the subcellular localization or targeting of the
protein; (7) alteration of the temporally regulated expression of
the protein (such that the protein is expressed when it normally
would not be, or alternatively is not expressed when it normally
would be); and (8) alteration of the localized (e.g., organ or
tissue specific) expression of the protein (such that the protein
is not expressed where it would normally be expressed or is
expressed where it normally would not be expressed), each compared
to a control or standard.
[0081] One particular type of protein abnormality that is
contemplated herein involves changes in the post-translational
processing of a protein. A protein that is abnormally
post-translationally processed includes, for instance, proteins
that processed in a different way, or to a different extent, than
the wild-type (normal) version of the protein. Progerin is an
example of an abnormally processed protein; it is not
proteolytically processed in the same way as the corresponding
wild-type protein, lamin A. In addition, progerin is abnormal in
that it remains farnesylated after its processing is completed (to
the extent that a cell expressing a nucleic acid encoding progerin
processes the protein). This can be referred to as being
"constitutively" farnesylated, in that an individual progerin
protein remains farnesylated in the context of an otherwise
unaltered cell. In contrast, a "normal" lamin A protein is only
transiently farnesylated, because full post-translational
processing of lamin A eventually results in loss of the farnesyl
group from the mature protein (by way of a required proteolytic
cleavage step). Progerin can be considered an abnormally
farnesylated form of lamin A; other abnormally farnesylated forms
of lamins are discussed herein, including forms engineered to lack
a proteolytic cleavage site, to lack the farnesylation site, and so
forth. Other potential "abnormal" post-translational processing
includes changes in other added groups (e.g., methylations,
phosphorylations, glycosylations, and so forth), alterations in
proteolytic processing, and so forth.
[0082] Controls or standards appropriate for comparison to a
sample, for the determination of abnormality, include samples
believed to be normal as well as laboratory determined values, even
though such values are possibly arbitrarily set, and keeping in
mind that such values may vary from laboratory to laboratory.
Laboratory standards and values may be set based on a known or
determined population value and may be supplied in the format of a
graph or table that permits easy comparison of measured,
experimentally determined values.
[0083] Analog, derivative or mimetic: An analog is a molecule that
differs in chemical structure from a parent or reference compound,
for example a homolog (differing by an incremental change in the
chemical structure, such as a difference in the length of an alkyl
chain), a molecular fragment, a structure that differs by one or
more functional groups, a change in ionization. Structural analogs
are often found using quantitative structure activity relationships
(QSAR), with techniques such as those disclosed in Remington (The
Science and Practice of Pharmacology, 19th Edition (1995), chapter
28). A derivative is a substance related to a base structure, and
theoretically derivable from the base structure. A mimetic is a
biomolecule that mimics the activity of another biologically active
molecule. Biologically active molecules can include chemical
structures that mimic the biological activities of a compound, for
instance a native siRNA.
[0084] Array: An arrangement of molecules, particularly biological
macromolecules (such as polypeptides or nucleic acids) in
addressable locations on a substrate. The array may be regular
(arranged in uniform rows and columns, for instance) or irregular.
The number of addressable locations on the array can vary, for
example from a few (such as three) to more than 50, 100, 200, 500,
1000, 10,000, or more. A "microarray" is an array that is
miniaturized so as to require or benefit from microscopic
examination, or other magnification, for its evaluation. Further
miniaturization can be used to produce "nanoarrays."
[0085] Within an array, each arrayed molecule sample is
addressable, in that its location can be reliably and consistently
determined within at least two dimensions of the array surface. In
ordered arrays, the location of each molecule sample can be
assigned to the sample at the time when it is spotted or otherwise
applied onto the array surface, and a key may be provided in order
to correlate each location with the appropriate target. Often,
ordered arrays are arranged in a symmetrical grid pattern, but
samples could be arranged in other patterns (e.g., in radially
distributed lines, spiral lines, or ordered clusters). Addressable
arrays are computer readable, in that a computer can be programmed
to correlate a particular address on the array with information
(such as hybridization or binding data, including for instance
signal intensity). In some examples of computer readable formats,
the individual "spots" on the array surface will be arranged
regularly in a pattern (e.g., a Cartesian grid pattern) that can be
correlated to address information by a computer.
[0086] The sample application "spot" on an array may assume many
different shapes. Thus, though the term "spot" is used, it refers
generally to a localized deposit of nucleic acid, and is not
limited to a round or substantially round region. For instance,
substantially square regions of mixture application can be used
with arrays encompassed herein, as can be regions that are
substantially rectangular (such as a slot blot-type application),
or triangular, oval, or irregular. The shape of the array substrate
itself is also immaterial, though it is usually substantially flat
and may be rectangular or square in general shape.
[0087] DNA (deoxyribonucleic acid): DNA is a long chain polymer
which comprises the genetic material of most living organisms (some
viruses have genes comprising ribonucleic acid (RNA)). The
repeating units in DNA polymers are four different nucleotides,
each of which comprises one of the four bases, adenine, guanine,
cytosine and thymine bound to a deoxyribose sugar to which a
phosphate group is attached. Triplets of nucleotides (referred to
as codons) code for each amino acid in a polypeptide, or for a stop
signal. The term codon is also used for the corresponding (and
complementary) sequences of three nucleotides in the mRNA into
which the DNA sequence is transcribed.
[0088] Unless otherwise specified, any reference to a DNA molecule
is intended to include the reverse complement of that DNA molecule.
Except where single-strandedness is required by the text herein,
DNA molecules, though written to depict only a single strand,
encompass both strands of a double-stranded DNA molecule. Thus, a
reference to the nucleic acid molecule that encodes lamin A, or a
fragment thereof, encompasses both the sense strand and its reverse
complement. Thus, for instance, it is appropriate to generate
probes or primers from the reverse complement sequence of the
disclosed nucleic acid molecules.
[0089] Deletion: The removal of a sequence of DNA, the regions on
either side of the removed sequence being joined together.
[0090] Epitope tags are short stretches of amino acids to which a
specific antibody can be raised, which in some embodiments allows
one to specifically identify and track the tagged protein that has
been added to a living organism or to cultured cells. Detection of
the tagged molecule can be achieved using a number of different
techniques. Examples of such techniques include:
immunohistochemistry, immunoprecipitation, flow cytometry,
immunofluorescence microscopy, ELISA, immunoblotting ("western"),
and affinity chromatography. Examples of useful epitope tags
include but are not limited to FLAG, T7, HA (hemagglutinin) and
myc.
[0091] Feature: An addressable spot/element containing a molecule
or mixture of molecules on an array. Features may be created by
printing the molecule(s), usually within some type of matrix, onto
the array platform by a printing device, such as a quill-like pen,
or by a touch-less deposition system (see, e.g., Harris et al.,
Nature Biotech. 18:384-385, 2000). Alternatively, in some
embodiments the feature can be made by in situ synthesis of the
molecule on the array substrate.
[0092] Fluorescent protein: A protein that either directly (through
its primary, secondary, or tertiary structure) or indirectly
(through a co-factor, non-protein chromophore, or a substrate, or
due to the addition of a fluor) produces or emits fluorescent
light. Non-limiting examples of fluorescent proteins are the green
fluorescent protein (GFP; see, for instance, GenBank Accession
Number M62654) from the Pacific Northwest jellyfish, Aequorea
victoria and natural and engineered variants thereof (see, for
instance, U.S. Pat. Nos. 5,804,387; 6,090,919; 6,096,865;
6,054,321; 5,625,048; 5,874,304; 5,777,079; 5,968,750; 6,020,192;
and 6,146,826; and published international patent application WO
99/64592).
[0093] Fluorophore: A chemical compound, which when excited by
exposure to a particular wavelength of light, emits light (i.e.,
fluoresces), for example at a different wavelength. Fluorophores
can be described in terms of their emission profile, or "color."
Green fluorophores, for exampleCY3.RTM. green fluorophore, FITC,
and OREGON GREEN.RTM. green fluorophore, are characterized by their
emission at wavelengths generally in the range of 515-540.lamda..
Red fluorophores, for example TEXAS RED.RTM. red fluorophore,
CY5.RTM. red fluorophore and tetramethylrhodamine, are
characterized by their emission at wavelengths generally in the
range of 590-690.lamda..
[0094] Examples of fluorophores that may be used are provided in
U.S. Pat. No. 5,866,366 to Nazarenko et al., and include for
instance: 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic
acid, acridine and derivatives such as acridine and acridine
isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid
(EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5
disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide,
anthranilamide, Brilliant Yellow, coumarin and derivatives such as
coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;
4',6-diaminidino-2-phenylindole (DAPI);
5',5''-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl
chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL);
4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin
and derivatives such as eosin and eosin isothiocyanate; erythrosin
and derivatives such as erythrosin B and erythrosin isothiocyanate;
ethidium; fluorescein and derivatives such as 5-carboxyfluorescein
(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC);
fluorescamine; IR144; IR1446; Malachite Green isothiocyanate;
4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine;
pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde;
pyrene and derivatives such as pyrene, pyrene butyrate and
succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron.RTM.
Brilliant Red 3B-A); rhodamine and derivatives such as
6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine
rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B,
rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B,
sulforhodamine 101 and sulfonyl chloride derivative of
sulforhodamine 101 (TEXAS RED.RTM. red fluorophore);
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl
rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);
riboflavin; rosolic acid and terbium chelate derivatives.
[0095] Other contemplated fluorophores include GFP (green
fluorescent protein), Lissamine.TM., diethylaminocoumarin,
fluorescein chlorotriazinyl, naphthofluorescein,
4,7-dichlororhodamine and xanthene and derivatives thereof. Other
fluorophores known to those skilled in the art may also be
used.
[0096] Inhibiting or treating a disease: Inhibiting the full
development of a disease, disorder or condition, for example, in a
subject who is at risk for a disease such as a laminopathy, an
aging-associated disease or condition, atherosclerosis or
cardiovascular disease. "Treatment" refers to a therapeutic
intervention that ameliorates a sign or symptom of a disease or
pathological condition after it has begun to develop. As used
herein, the term "ameliorating," with reference to a disease,
pathological condition or symptom, refers to any observable
beneficial effect of the treatment. The beneficial effect can be
evidenced, for example, by a delayed onset of clinical symptoms of
the disease in a susceptible subject, a reduction in severity of
some or all clinical symptoms of the disease, a slower progression
of the disease, a reduction in the number of relapses of the
disease, an improvement in the overall health or well-being of the
subject, or by other parameters well known in the art that are
specific to the particular disease or condition.
[0097] Injectable composition: A pharmaceutically acceptable fluid
composition including at least one active ingredient (for instance,
a dsRNA). The active ingredient is usually dissolved or suspended
in a physiologically acceptable carrier, and the composition can
additionally include minor amounts of one or more non-toxic
auxiliary substances, such as emulsifying agents, preservatives,
and pH buffering agents and the like. Such injectable compositions
that are useful for use with the provided nucleotides and proteins
are conventional; appropriate formulations are well known in the
art.
[0098] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, protein or organelle) has been substantially
separated or purified away from other biological components in the
cell of the organism in which the component naturally occurs, e.g.,
other chromosomal and extra-chromosomal DNA and RNA, proteins and
organelles. Nucleic acids and proteins that have been "isolated"
include nucleic acids and proteins purified by standard
purification methods. The term also embraces nucleic acids and
proteins prepared by recombinant expression in a host cell as well
as chemically synthesized nucleic acids.
[0099] Label: A composition detectable by (for instance)
spectroscopic, photochemical, biochemical, immunochemical, or
chemical means. Typical labels include fluorescent proteins or
protein tags, fluorophores, radioactive isotopes (including for
instance .sup.32P), ligands, biotin, digoxigenin, chemiluminescent
agents, electron-dense reagents (such as metal sols and colloids),
and enzymes (e.g., for use in an ELISA), haptens, and proteins or
peptides (such as epitope tags) for which antisera or monoclonal
antibodies are available. Methods for labeling and guidance in the
choice of labels useful for various purposes are discussed, e.g.,
in Sambrook et al., in Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (1989) and Ausubel et al., in
Current Protocols in Molecular Biology, John Wiley & Sons, New
York (1998). A label often generates a measurable signal, such as
radioactivity, fluorescent light or enzyme activity, which can be
used to detect and/or quantitate the amount of labeled
molecule.
[0100] Nucleotide: "Nucleotide" includes, but is not limited to, a
monomer that includes a base linked to a sugar, such as a
pyrimidine, purine or synthetic analogs thereof, or a base linked
to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide
is one monomer in a polynucleotide. A nucleotide sequence refers to
the sequence of bases in a polynucleotide.
[0101] Oligonucleotide: An oligonucleotide is a plurality of joined
nucleotides joined by native phosphodiester bonds, between about 6
and about 300 nucleotides in length. An oligonucleotide analog
refers to moieties that function similarly to oligonucleotides but
have non-naturally occurring portions. For example, oligonucleotide
analogs can contain non-naturally occurring portions, such as
altered sugar moieties or inter-sugar linkages, such as a
phosphorothioate oligodeoxynucleotide. Functional analogs of
naturally occurring polynucleotides can bind to RNA or DNA, and
include peptide nucleic acid (PNA) molecules.
[0102] Particular oligonucleotides and oligonucleotide analogs can
include linear sequences up to about 200 nucleotides in length, for
example a sequence (such as DNA or RNA) that is at least 6 bases,
for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or
even 200 bases long, or from about 6 to about 50 bases, for example
about 10-25 bases, such as 12, 15 or 20 bases.
[0103] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein-coding regions, in the same reading frame.
[0104] Open reading frame: A series of nucleotide triplets (codons)
coding for amino acids without any internal termination codons.
These sequences are usually translatable into a peptide.
[0105] Parenteral: Administered outside of the intestine, e.g., not
via the alimentary tract. Generally, parenteral formulations are
those that will be administered through any possible mode except
ingestion. This term especially refers to injections, whether
administered intravenously, intrathecally, intramuscularly,
intraperitoneally, or subcutaneously, and various surface
applications including intranasal, intradermal, and topical
application, for instance.
[0106] Peptide Nucleic Acid (PNA): An oligonucleotide analog with a
backbone comprised of monomers coupled by amide (peptide) bonds,
such as amino acid monomers joined by peptide bonds.
[0107] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful with this disclosure are conventional.
Martin, Remington's Pharmaceutical Sciences, published by Mack
Publishing Co., Easton, Pa., 19th Edition, 1995, describes
compositions and formulations suitable for pharmaceutical delivery
of the nucleotides and proteins herein disclosed.
[0108] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0109] Polymorphism: Variant in a sequence of a gene. Polymorphisms
can be those variations (nucleotide sequence differences) that,
while having a different nucleotide sequence, produce functionally
equivalent gene products, such as those variations generally found
between individuals, different ethnic groups, geographic locations.
The term polymorphism also encompasses variations that produce gene
products with altered function, e.g., variants in the gene sequence
that lead to gene products that are not functionally equivalent.
This term also encompasses variations that produce no gene product,
an inactive gene product, or increased gene product. The term
polymorphism may be used interchangeably with allele or mutation,
unless context clearly dictates otherwise.
[0110] Polymorphisms can be referred to, for instance, by the
nucleotide position at which the variation exists, by the change in
amino acid sequence caused by the nucleotide variation, or by a
change in some other characteristic of the nucleic acid molecule
that is linked to the variation (e.g., an alteration of a secondary
structure such as a stem-loop, or an alteration of the binding
affinity of the nucleic acid for associated molecules, such as
polymerases, RNases, and so forth). In the current instance,
Mutation 1 is also referred to as G608G(GGC>GGT), indicating
that the mutation is in codon 608, that it is silent (in that it
causes no change in the encoded amino acid), and that the exact
nucleotide sequence change is C to T in the third position of the
codon. Similarly, Mutation 2 is also referred to as
G608S(GGC>AGC), indicating that the mutation is in codon 608,
that it causes an amino acid substitution (glycine to serine), and
that the exact nucleotide sequence change is G to A in the first
position of the codon.
[0111] Probes and primers: A probe comprises an isolated nucleic
acid attached to a detectable label or other reporter molecule.
Typical labels include radioactive isotopes, enzyme substrates,
co-factors, ligands, chemiluminescent or fluorescent agents,
haptens, and enzymes. Methods for labeling and guidance in the
choice of labels appropriate for various purposes are discussed,
e.g., in Sambrook et al. (In Molecular Cloning: A Laboratory
Manual, CSHL, New York, 1989) and Ausubel et al. (In Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
1998).
[0112] Primers are short nucleic acid molecules, for instance DNA
oligonucleotides 10 nucleotides or more in length, for example that
hybridize to contiguous complementary nucleotides or a sequence to
be amplified. Longer DNA oligonucleotides may be about 15, 20, 25,
30 or 50 nucleotides or more in length. Primers can be annealed to
a complementary target DNA strand by nucleic acid hybridization to
form a hybrid between the primer and the target DNA strand, and
then the primer extended along the target DNA strand by a DNA
polymerase enzyme. Primer pairs can be used for amplification of a
nucleic acid sequence, e.g., by the polymerase chain reaction (PCR)
or other nucleic-acid amplification methods known in the art. Other
examples of amplification include strand displacement
amplification, as disclosed in U.S. Pat. No. 5,744,311;
transcription-free isothermal amplification, as disclosed in U.S.
Pat. No. 6,033,881; repair chain reaction amplification, as
disclosed in WO 90/01069; ligase chain reaction amplification, as
disclosed in EP-A-320 308; gap filling ligase chain reaction
amplification, as disclosed in U.S. Pat. No. 5,427,930; and
NASBA.TM. RNA transcription-free amplification, as disclosed in
U.S. Pat. No. 6,025,134.
[0113] Nucleic acid probes and primers can be readily prepared
based on the nucleic acid molecules provided in this disclosure. It
is also appropriate to generate probes and primers based on
fragments or portions of these disclosed nucleic acid molecules,
for instance regions that encompass the identified polymorphisms at
nucleotide 1822 and nucleotide 1824 within the LMNA coding
sequence.
[0114] Methods for preparing and using nucleic acid probes and
primers are described, for example, in Sambrook et al. (In
Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989),
Ausubel et al. (ed.) (In Current Protocols in Molecular Biology,
John Wiley & Sons, New York, 1998), and Innis et al. (PCR
Protocols, A Guide to Methods and Applications, Academic Press,
Inc., San Diego, Calif., 1990). Amplification primer pairs can be
derived from a known sequence, for example, by using computer
programs intended for that purpose such as Primer (Version 0.5,
.COPYRGT. 1991, Whitehead Institute for Biomedical Research,
Cambridge, Mass.). One of ordinary skill in the art will appreciate
that the specificity of a particular probe or primer increases with
its length. Thus, for example, a primer comprising 30 consecutive
nucleotides of a lamin A-encoding nucleotide or flanking region
thereof (a "lamin A primer" or "lamin A probe") will anneal to a
target sequence with a higher specificity than a corresponding
primer of only 15 nucleotides. Thus, in order to obtain greater
specificity, probes and primers can be selected that comprise at
least 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of
a lamin A nucleotide sequences.
[0115] The disclosure thus includes isolated nucleic acid molecules
that comprise specified lengths of the lamin A encoding sequence
and/or flanking regions. Such molecules may comprise at least about
10, 15, 20, 23, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of
these sequences or more, and may be obtained from any region of the
disclosed sequences. By way of example, the human LMNA locus, cDNA,
ORF, coding sequence and gene sequences (including sequences both
upstream and downstream of the LMNA coding sequence) may be
apportioned into about halves or quarters based on sequence length,
and the isolated nucleic acid molecules (e.g., oligonucleotides)
may be derived from the first or second halves of the molecules, or
any of the four quarters. The cDNA also could be divided into
smaller regions, e.g. about eighths, sixteenths, twentieths,
fiftieths and so forth, with similar effect.
[0116] In particular embodiments, isolated nucleic acid molecules
comprise or overlap at least one residue position designated as
being associated with a polymorphism that is predictive of progeria
and/or a premature aging disease or condition. Such polymorphism
sites include position 1822 (corresponding to the Mutation 2
polymorphism) and position 1824 (corresponding to the Mutation 1
polymorphism).
[0117] Protein: A biological molecule, particularly a polypeptide,
expressed by a gene and comprised of amino acids.
[0118] Purified: The term "purified" does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified protein preparation is one in which the protein
referred to is more pure than the protein in its natural
environment within a cell or within a production reaction chamber
(as appropriate).
[0119] Recombinant: A recombinant nucleic acid is one that has a
sequence that is not naturally occurring or has a sequence that is
made by an artificial combination of two otherwise separated
segments of sequence. This artificial combination can be
accomplished by chemical synthesis or, more commonly, by the
artificial manipulation of isolated segments of nucleic acids,
e.g., by genetic engineering techniques.
[0120] Specific binding agent: An agent that binds substantially
only to a defined target. Thus a lamin A protein-specific binding
agent binds substantially only the lamin A protein. As used herein,
the term "Lamin protein specific binding agent" includes anti-Lamin
protein antibodies (and functional fragments thereof) and other
agents (such as soluble receptors) that bind substantially only to
a Lamin protein. It is particularly contemplated in specific
embodiments that certain Lamin-specific binding agents are specific
for one form of Lamin, such as lamin A or lamin C.
[0121] Anti-Lamin protein antibodies may be produced using standard
procedures described in a number of texts, including Harlow and
Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). The
determination that a particular agent binds substantially only to
the target protein may readily be made by using or adapting routine
procedures. One suitable in vitro assay makes use of the Western
blotting procedure (described in many standard texts, including
Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York,
1988)). Western blotting may be used to determine that a given
target protein binding agent, such as an anti-lamin A protein
monoclonal antibody, binds substantially only to the specified
target protein.
[0122] Shorter fragments of antibodies can also serve as specific
binding agents. For instance, FAbs, Fvs, and single-chain Fvs
(SCFvs) that bind to lamin A would be lamin A-specific binding
agents. These antibody fragments are defined as follows: (1) FAb,
the fragment which contains a monovalent antigen-binding fragment
of an antibody molecule produced by digestion of whole antibody
with the enzyme papain to yield an intact light chain and a portion
of one heavy chain; (2) FAb', the fragment of an antibody molecule
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain; two FAb' fragments are obtained per antibody molecule;
(3) (FAb').sub.2, the fragment of the antibody obtained by treating
whole antibody with the enzyme pepsin without subsequent reduction;
(4) F(Ab').sub.2, a dimer of two FAb' fragments held together by
two disulfide bonds; (5) Fv, a genetically engineered fragment
containing the variable region of the light chain and the variable
region of the heavy chain expressed as two chains; and (6) single
chain antibody ("SCA"), a genetically engineered molecule
containing the variable region of the light chain, the variable
region of the heavy chain, linked by a suitable polypeptide linker
as a genetically fused single chain molecule. Methods of making
these fragments are routine.
[0123] Subject: Living multi-cellular vertebrate organisms, a
category that includes both human and non-human mammals. This term
encompasses both known and unknown individuals, such that there is
no requirement that a person working with a sample from a subject
know who the subject is, or even from where the sample was
acquired.
[0124] Subject susceptible to a disease or condition: A subject
capable of, prone to, or predisposed to developing a disease or
condition. It is understood that a subject already having or
showing symptoms of a disease or condition is considered
"susceptible" since they have already developed it.
[0125] Therapeutically effective dose: A dose sufficient to prevent
advancement, or to cause regression of the disease, or which is
capable of relieving symptoms caused by the disease, such as a
laminopathy or age related disease or condition (for instance,
atherosclerosis). For example, a therapeutically effective amount
of an inhibitor can vary from about 0.1 nM per kilogram (kg) body
weight to about 1 .mu.M per kg body weight, such as about 1 nM to
about 500 nM per kg body weight, or about 5 nM to about 50 nM per
kg body weight. The exact dose is readily determined by one of
skill in the art based on the potency of the specific compound, the
age, weight, sex and physiological condition of the subject.
[0126] Transfected or Transformed: A process by which a nucleic
acid molecule is introduced into live cells, for instance by
molecular biology techniques, resulting in a transfected (or
transformed) cell. As used herein, the term transfection
encompasses all techniques by which a nucleic acid molecule might
be introduced into such a cell, including transduction with viral
vectors, transfection with plasmid vectors, and introduction of DNA
by electroporation, lipofection, and particle gun acceleration.
[0127] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transfected host cell. Recombinant DNA
vectors are vectors having recombinant DNA. A vector can include
nucleic acid sequences that permit it to replicate in a host cell,
such as an origin of replication. A vector can also include one or
more selectable marker genes and other genetic elements known in
the art. Viral vectors are recombinant DNA vectors having at least
some nucleic acid sequences derived from one or more viruses.
[0128] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Hence "comprising A or B" means including A,
or B, or A and B. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including explanations of terms, will
control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
III. Overview of Several Embodiments
[0129] A first embodiment is a method of reducing at least one
cellular defect in a cell from a subject susceptible to a disease
or condition characterized by farnesylation of an abnormally
farnesylated form of a lamin, comprising administering to the cell
a therapeutically effective dose of a farnesyltransferase inhibitor
(FTI). In examples of such methods, the cellular defect involves
mis-localization of a farnesylated lamin, mis-localization of a
non-farnesylated lamin, nuclear membrane disruption, aggregation of
lamin, nuclear lobulation, nuclear blebbing, cytoskeleton
disruption, early senescence, apoptosis, reduced secretion of
MMP-3, or two or more thereof.
[0130] Examples of diseases or conditions characterized by
farnesylation of an abnormally farnesylated form of a lamin include
laminopathies, which include but are not limited to
Hutchinson-Gilford Progeria Syndrome (HGPS), progeria, muscular
dystrophy, Charcot-Marie-Tooth disorder, and Werner syndrome.
[0131] There is also provided a method of treating a subject having
or predisposed to a laminopathy, which method involves
administering to the subject an effective amount of at least one
FTI.
[0132] Yet another provided method is a method of preventing
progression of a laminopathy in a subject having or predisposed to
the laminopathy, which method involves administering at least one
FTI to the subject.
[0133] In examples of the various embodiments, the normally
non-farnesylated lamin is a lamin other than Lamin B. For instance,
in specific instances, the normally non-farnesylated lamin is lamin
A.
[0134] It is specifically contemplated herein that the subject of
any of the provided methods may be a subject having, or suspected
of having, or susceptible to, atherosclerosis.
[0135] By way of specific, non-limiting examples, the FTI used in
any method provided herein can be PD169541, R115777, SCH66336, or a
farnesyltransferase inhibitory derivative thereof. Optionally, in
any of the methods, a second therapeutic compound can be
administered to the cell or subject. Examples of such second
therapeutic compounds include another FTI, or a non-FTI
compound.
[0136] A further embodiment is a method of screening for a compound
useful in treating a laminopathy in a mammal. In an example of this
embodiment, the method involves determining if a test compound
alters a cellular defect selected from the group consisting of
mis-localization of a farnesylated lamin, mis-localization of a
non-farnesylated lamin, nuclear membrane disruption, aggregation of
lamin, nuclear lobulation, nuclear blebbing, cytoskeleton
disruption, early senescence, apoptosis, reduced secretion of
MMP-3, or two or more thereof; and selecting a compound that alters
the cellular defect so that it is more close to normal.
[0137] Also provided is a method for identifying an agent useful
for treating a laminopathy, which method involves contacting the
agent to a cell expressing progerin or a progerin-like abnormal
lamin A protein under conditions sufficient to allow interaction
between the cell and the agent; evaluating an amount of a cellular
defect in the cell; and comparing the amount of the cellular defect
in the cell contacted with the agent to an amount of the cellular
defect in a control cell not treated with the agent, wherein a
statistically significant (e.g., p<0.05, or p<0.01, or
another selected level of significance) improvement in the amount
of the cellular defect in the cell contacted with the agent, as
compared to the control cell not treated with the agent, identifies
the agent as one that is useful for treating the laminopathy.
[0138] Yet a further method provided herein is a method for
identifying an agent that inhibits or reverses at least one
cellular defect caused by constitutive farnesylation of a lamin
protein. Such a method involves contacting a cell expressing
progerin or a progerin-like abnormal lamin A protein with a test
agent under conditions sufficient to allow interaction between the
cell and the agent; and determining whether a cellular defect
caused by constitutive farnesylation of a lamin protein is
inhibited or reversed. By way of non-limiting example, the cellular
defect is mis-localization of a farnesylated lamin,
mis-localization of a non-farnesylated lamin, nuclear membrane
disruption, aggregation of lamin, nuclear lobulation, nuclear
blebbing, cytoskeleton disruption, early senescence, apoptosis,
reduced secretion of MMP-3, or a combination of two or more
thereof.
IV. Diseases Linked to Lamin A/C
[0139] Over 180 mutations have been reported in the LMNA gene, and
currently there are eight diseases in addition to HGPS, referred to
as the "laminopathies," that are associated with various mutations
in this gene (Gruenbaum et al., Nat. Rev. Mol. Cell Biol. 6:21-31,
2005). These include such disorders as Emery-Dreifuss muscular
dystrophy (EDMD; e.g., associated with heterozygous R527P),
mandibuloacral dysplasia (associated with homozygous R527H),
atypical Werner's syndrome, dilated cardiomyopathy-type 1A (CMD1A;
e.g., associated with R644C), restrictive dermopathy, and
Dunnigan-type familial partial lipodystrophy (Mounkes et al., Curr.
Opin. Genet. Dev. 13:223-230, 2003; Navarro et al., Hum. Mol.
Genet. 13:2493-2503, 2004).
[0140] Emery-Dreifuss muscular dystrophy is an autosomal recessive
or dominant disease characterized by muscle weakness, contractures,
and cardiomyopathy with conduction defects. Familial partial
lipodystrophy (Dunnigan variety) (FPLD) is an autosomal dominant
disorder characterized by marked loss of subcutaneous adipose
tissue from the extremities and trunk but by excess fat deposition
in the head and neck. This condition is frequently associated with
profound insulin resistance, dyslipidemia, and diabetes.
[0141] Surprisingly, point mutations recently identified in the
LMNA gene have been found to cause HGPS (PCT/US2003/033058,
published as WO 04/035753, incorporated herein by reference in its
entirety). The inheritance is new mutation autosomal dominant, and
identified mutations occur in codon 608; the most common is due to
a C to T base substitution in a CpG dinucleotide. It is currently
believed that the mechanism of the mutations is activation of a
cryptic splice site within the LMNA gene, which leads to deletion
of part of exon 11 and generation of a lamin A protein product that
is 50 amino acids shorter than the normal protein. All of the
identified mutations are predicted to affect lamin A but not Lamin
C. In addition, two cases of classical HGPS were identified with
segmental UPD (uniparental disomy) of chromosome 1q from fibroblast
DNA do not show the mutation, which may be indicative of a (in vivo
or in vitro) somatic rescue event.
[0142] The results described herein can be generalized to the aging
process and related conditions and diseases, beyond progeroid
diseases. This is because HGPS is in many respects closely
connected to normal aging processes. HGPS continues to be
recognized as a useful model of aging (Fossel, J. Pediatr
Endocrinol Metab 13 Suppl 6:1477-1481, 2000). For instance, the
connection to atherosclerosis is very strong, especially of the
coronary arteries. In addition, alopecia in HGPS is similar to that
seen in subjects with advanced age. Further, the prime cellular
feature of HGPS, as described many years ago by Hayflick and others
(Hayflick, N Engl J Med 295:1302-1308, 1976) is early cellular
senescence. The limited number of cell divisions in HGPS
fibroblasts is similar to what is seen in fibroblasts derived from
elderly individuals. That was further explored by research showing
similarities in the gene expression patterns of HGPS fibroblasts
and those derived from elderly persons, distinguishing them from
fibroblasts derived from younger persons (Ly et al., Science 287:
2486-2492, 2000).
V. Specific HGPS Mutations Identified in LMNA
[0143] The typical LMNA mutation in HGPS is a nucleotide
substitution of a C to a T at position 1824 of SEQ ID NO: 19
(corresponding to position 4277 in GI 292250 (Accession No.
L12401)), causing no change in the encoded amino acid (G608G)
(Accession No. P02545), but creating a cryptic splice donor site.
Activation of this site results in an mRNA lacking 150 nucleotides.
This is in turn translated into a mutant protein with a 50 amino
acid internal deletion near the C-terminal end, termed progerin
(Eriksson et al., Nature 423:293-298, 2003). Lamin A is normally
expressed by most differentiated cells, where it integrally affects
both nuclear membrane structure and function (Rober et al.,
Development 105:365-378, 1989). Progerin apparently acts in a
dominant negative manner on the nuclear function of cell types that
express lamin A (Goldman et al., Proc. Natl. Acad. Sci. USA
101:8963-8968, 2004; Scaffidi & Misteli, Nat. Med. 11:440-445,
2005). In addition to the potential mechanical fragility created by
disrupting the nuclear lamina, this mutation also may affect other
vital cellular processes such as gene transcription, DNA
replication, and cell division.
[0144] There has also been identified a change at nucleotide
position 4275 in GI 292250, which corresponds to amino acid 608
(corresponding to position 1822 of SEQ ID NO: 19); this mutation
changes a glycine to a serine in lamin A, and is predicted to
generate the same cryptic splice site as the silent mutation. Hence
both mutations generate the same mutant lamin A protein (progerin).
The two mutations both occur in the same codon, which encodes amino
acid 608.
[0145] The discovery that rare variants in the sequence of LMNA
causes HGPS also enables a variety of diagnostic, prognostic, and
therapeutic methods that are further embodiments. The new
appreciation of the role of lamin A in HGPS and more generally
aging illnesses and arteriosclerosis/atherosclerosis enables
detection of predisposition to these conditions in a subject. This
disclosure also enables early detection of subjects at high risk of
these conditions, and provides opportunities for prevention and/or
early treatment.
[0146] The deletion of the last half of exon 11 removes a cleavage
site that is normally necessary for processing of lamin A.
Post-translational modification of the CAAX box (SEQ ID NO: 31) at
the C-terminus of lamin A, which is still present in the mutant
forms, allows anchoring of the protein in the membrane--but then
this anchoring mechanism is normally removed by the processing
cleavage. The lamin A mutant protein (progerin) shown herein is not
cleaved, and thus appears to be trapped in this membrane location.
Since lamin A is part of a large multiprotein complex, its
mislocalization is likely to pull other bystander proteins into the
same improper location. It is now believed that this leads to
structural abnormalities of the nucleus that can be visualized by
light microscopy, immunohistochemistry, immunofluorescence,
confocal microscopy, or electron microscopy. These characteristics
are also useful for providing systems to test compounds for
treating laminopathies.
VI. Farnesyltransferase Inhibitors to Ameliorate Progerin Cellular
Disruption
[0147] Farnesylation is a permanent posttranslational modification;
therefore, proteolytic cleavage is required to remove the
farnesylated residue from lamin A protein and thereby effect
defarnesylation and maturation of fully processed lamin A. Although
it can be farnesylated, the progerin protein cannot be
defarnesylated because it is missing the second cleavage site
necessary for binding the protease ZMPSTE24, whose cleavage
activity removes the last 15 amino acids in the C-terminal portion
of the protein and effects defarnesylation (see, for instance,
Eriksson et al., Nature 423(6937):293-298, 2003, incorporated
herein by reference). Progerin maintains its nuclear localization
sequence and its farnesyl group, along with the central rod that
allows dimerization with normal lamin A and probably with itself.
The result is an aberrant farnesylated protein putatively capable
of altering normal lamin A function as a dominant negative, as well
as assuming its own aberrant function through its association with
the nuclear membrane. Thus, the multisystem disease process and the
variety of genes that are affected in HGPS (Csoka et al., Aging
Cell 3:235-243, 2004) lie downstream of a protein defect that is
central to basic cellular function.
[0148] The progerin deletion has a number of consequences which
give rise to the abnormal cellular phenotype, including abnormal
protein processing and deletion of critical nuclear periphery
protein binding domains. The retention of the farnesyl group
changes important properties of the protein making it more
lipophilic and altering interactions with other proteins. We have
demonstrated in vivo that progerin is incompletely processed and
retains the farnesyl group (Example 1). Expression of an N-terminal
GFP-progerin fusion protein in normal fibroblasts caused abnormal
nuclear morphology similar to that seen on HGPS fibroblasts.
Furthermore, expression of the endogenous mutant in HGPS
fibroblasts can cause abnormal localization of normal GFP tagged
lamin A, demonstrating a dominant effect of the mutant protein on
exogenously expressed normal lamin A. However, expression of the
normal GFP-lamin A did not improve the nuclear morphology phenotype
of HGPS fibroblasts, indicating that the deleterious effects of
progerin are not compensated by increased expression of the normal
protein.
[0149] It is also demonstrated herein that a protein cleavage site
mutation construct gives rise to cellular phenotypes similar to a
progerin construct (Example 2). A GFP-tagged cleavage-minus mutant
lamin A (L647R) displayed a localization pattern very similar to
the localization of GFP-tagged progerin when expressed in normal
fibroblasts. Its expression caused a significant increase in
abnormal nuclear morphology similar to the effect of the
GFP-progerin construct.
[0150] Treatment of normal fibroblasts expressing progerin and HGPS
fibroblasts with the farnesyltransferase inhibitor PD 169541
resulted in a redistribution of GFP-lamin A and GFP-progerin
localization and significant improvement in the nuclear morphology
phenotype (Example 3). These studies indicate that abnormal
farnesylation of progerin plays a major role in the abnormal
cellular phenotype and suggest a possible therapeutic option for
HGPS.
[0151] Based on the discoveries presented herein, it is shown that
farnesyltransferase inhibitors (both direct effectors and indirect
inhibitors) will inhibit or reduce the formation of progerin, cause
a decrease in lamin A protein and an increase prelamin A protein.
Decreasing the amount of aberrant protein may improve disease
status in progeria and other laminopathies; this is supported by
the discovery that treatment with an FTI reduces cellular defects
linked to the expression of progerin. It is therefore believed that
a farnesyltransferase inhibitor, such as (but not limited to)
PD169541, R115777 (tipifarnib, Zarnestra), and SCH66336
(lonafarnib, Sarasar), will reduce or abolish the formation of
progerin, decrease lamin A, and increase the levels of a prelamin A
protein lacking 50 amino acids ("preprogerin"). This is
demonstrated herein (Example 3), where normal fibroblasts
expressing progerin and HGPS fibroblasts show a redistribution of
GFP-lamin A and GFP-progerin localization and significant
improvement in the nuclear morphology phenotype when exposed to the
farnesyltransferase inhibitor PD169541.
[0152] Farnesyltransferase inhibitors can be used to reverse and/or
prevent cellular effects caused by accumulation of progerin or
other forms of constitutively farnesylated lamin protein. Thus, it
is now determined that FTIs can be used to treat effects associated
with expression of constitutively farnesylated lamin A, including
for instance symptoms characteristic of HGPS, other laminopathies,
aging-related conditions, atherosclerosis, and so forth. Specific
cellular symptoms include mis-localization of farnesylated lamin,
such a form of a lamin A protein (including prelamin A, progerin,
preprogerin, and so forth), mis-localization of a non-farnesylated
lamin, such as one or more non-farnesylated lamin A protein form
(for instance, fully processed lamin A protein that is mislocalized
due to association with a farnesylated lamin protein form),
aggregation of lamin(s), nuclear membrane disruption, nuclear
blebbing, nuclear lobulation, early senescence, apoptosis, and
reduced secretion of MMP-3. In many instances, a cell will display
a combination of two or more of these symptoms/effects.
VII. Farnesyltransferase Inhibitors
[0153] Farnesyltransferase inhibitors (FTIs) were developed
originally as inhibitors of Ras biological activity, because FTase
modification of Ras by farnesylation is essential for Ras to cause
oncogenesis, and more generally as target-molecule specific
chemotherapeutic agents. FTIs inhibit the posttranslational
addition of a 15-carbon farnesyl group to a C-terminal cysteine
residue that is required, for instance, for farnesylated proteins
(e.g., Ras) to localize to the cell membrane (Reuter et al., Blood
96(5):1655-1669, 2000). FTIs effectively block signaling and
cellular transformation of some but not all isoforms of Ras,
although it is certain that Ras is not the only target of
farnesyltransferase inhibition (Ashar et al., J. Biol. Chem.
275(39):30451-30457, 2000; Liu et al., Mol. Cell Biol.
20(16):6105-6113, 2000). More generally, farnesyltransferase
inhibitors were described as potentially useful to treat cancers
where mutated forms of Ras are commonly found (e.g., colon and
pancreatic cancers). However, since the most common forms of Ras
found mutated in human cancers (K-Ras and N-Ras) undergo
alternative prenylation by GGTase I when cells are treated with FTI
(Whyte et al., J. Biol. Chem. 272:14459-14464, 1997), the
anti-tumor activity of FTIs is not attributed to blocking Ras
itself. Alternatively prenylated Ras, for instance
geranylgeranylated K-Ras, retains function and is not inhibited by
FTI. Instead, other substrates of FTase are believed to account for
the clinical activity of FTIs. Farnesylated proteins that become
alternatively prenylated in the presence of FTIs can escape the
inhibitory effects of these compounds and remain functional.
Therefore, it is critical to evaluate the possibility of
alternative prenylation when determining whether a particular
protein, for example progerin, can be targeted successfully by FTI
treatment. Clinical consideration and prospects for FTIs in general
are discussed in Cox and Der (Curr. Op. Pharma. 2:388-393,
2002).
[0154] FTIs generally can be divided into three groups:
tetrapeptides having or mimicking the CAAX motif (SEQ ID NO: 31)
(Brown et al., Proc. Natl. Acad. Sci. U.S.A. 89:8313-8316, 1992;
Reiss et al., Proc. Natl. Acad. Sci. U.S.A. 88:732-736, 1991;
Goldstein et al., J. Biol. Chem. 266:15575-15578, 1991); analogs of
farnesyl pyrophosphate (FPP) (Gibbs et al., J. Biol. Chem
268:7617-7620, 1993); and inhibitors with structures not resembling
either tetrapeptides or FPP (Liu et al., J. Antibiot. 45:454-457,
1992; Miura et al., FEBS Lett. 318:88-90, 1993; Omura et al., J.
Antibiot. 46:222-228, 1993; Van Der Pyl et al. J. Antibiot.
45:1802-1805, 1992). The latter category of inhibitors generally
has lower activity compared to the first two categories. By way of
example, the FTI SCH66336 is a non-peptidomimetic FTI; FTI-277 is a
peptidomimetic.
[0155] Several FTIs have entered into clinical trials in the last
several years. For instance, the clinical candidate FTI SCH66336
(lonafarnib, Sarasar.RTM.) has been shown to inhibit proliferation
of several human cancer cell lines and is active against human
brain, lung, prostate, pancreas, colon, and bladder tumor
xenografts in nude mice (Liu et al., Cancer Res. 58(21):4947-4956,
1998; Feldkamp et al., Cancer Res., 61(11):4425-4431, 2001). One
Phase I clinical trial showed that SCH66336 inhibits protein
farnesylation in vivo, and is generally well tolerated by the
subject (Adjei et al., Cancer Res., 60(7):1871-1877, 2000).
Anti-leukemic activity of SCH66336 on cell culture and mouse models
of BCR/ABL has been demonstrated (Reichert et al., Blood,
97(5):1399-1403; 2001; Peters et al., Blood, 97(5):1404-1412,
2001).
[0156] The FTI R115777 (tipifarnib, Zarnestra.RTM.) has also
entered clinical trials; it has been found to be well tolerated,
but not fully effective against cancer (Adjei et al., J Clin Oncol.
21(9):1760-1766, 2003; Heymach et al., Annals of Oncology
15:1187-1193, 2004).
[0157] It is shown herein that FTIs can be used to reverse and/or
prevent cellular effects caused by accumulation of progerin or
other forms of constitutively farnesylated lamin A. It is believed
that all categories of FTIs may be used in methods and compositions
provided herein; the selection of a specific FTI is within the
skill of the ordinary practitioner based on testing methods
provided herein. In some embodiments, it is beneficial to select an
inhibitor compound that is more selective for farnesyltransferase,
compared to geranylgeranyltransferase I. In other embodiments, it
may be beneficial to select an inhibitor compound that is dually
selective, in that it inhibits both FTase and GGTase I.
Considerations for determining selectivity criteria for FTIs
include (but are not limited to) the possibility of lower toxicity
with FTase-specific FTIs versus dual specificity FTIs, although
both efficacy and toxicity may differ according to the particular
compound and the particular patient. As will be recognized by an
ordinarily skilled practitioner, other considerations, for instance
pharmacological and medical considerations, may also apply.
[0158] The development and chemistry of FTIs are well documented
and known to those of ordinary skill By way of example, the
following publications review FTIs in the context of cancer
treatment: Cox & Der, Biochim Biophys Acta 1333:F51-F71, 1997;
Gelb et al., Curr Opin Chem Biol 2:40-48, 1998; Rowinsky et al., J.
Clin Oncol. 17; 3631-3652, 1999; Oliff, Biochim Biophys Acta
1423:C19-C30, 1999; Sebti & Hamilton, Expert Opin Investig
Drugs 9:2767-2782, 2000; and Gibbs et al., Curr Med Chem
8:1437-1465, 2001.
[0159] The following patent documents provide additional
descriptions of making example farnesyltransferase inhibitors and
compositions contain such compounds: WO 00/39082; WO 00/12499; WO
00/12498; WO 00/01691; WO 99/45912; WO 98/49157; WO 98/40383; WO
98/28303; WO 97/30992; WO 97/21701; WO 97/16443; WO 94/10138; U.S.
Application Publication 20040063770; U.S. Application Publication
20030060450; U.S. Pat. No. 6,187,786; U.S. Pat. No. 6,177,432; U.S.
Pat. No. 6,169,096; U.S. Pat. No. 6,133,303; U.S. Pat. No.
6,037,350; U.S. Pat. No. 5,976,851; U.S. Pat. No. 5,972,984; U.S.
Pat. No. 5,972,966; U.S. Pat. No. 5,968,965; U.S. Pat. No.
5,968,952; U.S. Pat. No. 5,965,578; U.S. Pat. No. 5,965,539; U.S.
Pat. No. 5,958,939; U.S. Pat. No. 5,939,557; U.S. Pat. No.
5,936,097; U.S. Pat. No. 5,891,889; U.S. Pat. No. 5,889,053; U.S.
Pat. No. 5,880,140; U.S. Pat. No. 5,872,135; U.S. Pat. No.
5,869,682; U.S. Pat. No. 5,861,529; U.S. Pat. No. 5,859,015; U.S.
Pat. No. 5,856,439; U.S. Pat. No. 5,856,326; U.S. Pat. No.
5,852,010; U.S. Pat. No. 5,843,941; U.S. Pat. No. 5,807,852; U.S.
Pat. No. 5,780,492; U.S. Pat. No. 5,773,455; U.S. Pat. No.
5,767,274; U.S. Pat. No. 5,756,528; U.S. Pat. No. 5,750,567; U.S.
Pat. No. 5,721,236; U.S. Pat. No. 5,700,806; U.S. Pat. No.
5,661,161; U.S. Pat. No. 5,602,098; U.S. Pat. No. 5,585,359; U.S.
Pat. No. 5,578,629; U.S. Pat. No. 5,534,537; U.S. Pat. No.
5,532,359; U.S. Pat. No. 5,523,430; U.S. Pat. No. 5,504,212; U.S.
Pat. No. 5,491,164; U.S. Pat. No. 5,420,245; and U.S. Pat. No.
5,238,922.
[0160] Also useful are pharmaceutically acceptable acid or base
addition salts of FTIs, including any of the inhibitors as
mentioned above. The phrase "pharmaceutically acceptable acid or
base addition salts" includes therapeutically active non-toxic acid
and non-toxic base addition salt forms which FTIs are able to form.
Such compounds which have basic properties can be converted in
their pharmaceutically acceptable acid addition salts by treating
said base form with an appropriate acid. Appropriate acids
comprise, for example, inorganic acids such as hydrohalic acids,
e.g. hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric
and the like acids; or organic acids such as, for example, acetic,
propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic,
succinic (i.e. butanedioic acid), maleic, fumaric, malic, tartaric,
citric, methanesulfonic, ethanesulfonic, benzenesulfonic,
p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic
and the like acids.
[0161] FTI compounds which have acidic properties may be converted
in their pharmaceutically acceptable base addition salts by
treating said acid form with a suitable organic or inorganic base.
Appropriate base salt forms comprise, for example, the ammonium
salts, the alkali and earth alkaline metal salts, e.g. the lithium,
sodium, potassium, magnesium, calcium salts and the like, salts
with organic bases, e.g. the benzathine, N-methyl-D-glucamine,
hydrabamine salts, and salts with amino acids such as, for example,
arginine, lysine and the like.
[0162] The terms acid or base addition salt also comprise the
hydrates and the solvent addition forms that an FTI is able to
form. Examples of such forms are, for instance, hydrates,
alcoholates and the like.
[0163] Also contemplated for use in methods and compositions
described herein are stereochemically isomeric forms of FTIs. The
term stereochemically isomeric form includes all possible compounds
made up of the same atoms bonded by the same sequence of bonds, but
having different three-dimensional structures that are not
interchangeable. Unless otherwise mentioned or indicated, the
chemical designation of a compound encompasses the mixture of all
possible stereochemically isomeric forms that the compound may
possess. Such mixture may contain all diastereomers and/or
enantiomers of the basic molecular structure of the compound. Also
contemplated are all stereochemically isomeric forms in pure form
or in admixture with each other. Also contemplated are tautomeric
forms of FTI compounds.
VIII. Methods of Treatment
[0164] The present disclosure includes methods of using various
compounds and compositions, including any of the listed FTIs or
other compounds identified as capable of inhibiting, directly or
indirectly, farnesylation of lamin A or progerin (referred to
generally as FTI compounds), as a treatment for disease. In a
particular embodiment, the FTI compound is a peptidomimetic or
non-peptidomimetic inhibitor of the farnesyltransferase enzyme, and
the treatment is a treatment of a laminopathy, such as HGPS or
another progeroid disease or condition, an age-related condition, a
cardiovascular disease or condition (such as atherosclerosis), and
so forth.
[0165] The method includes administering a pharmaceutical agent or
FTI, or more than one, or a combination of a FTI (or more than one)
and one or more other pharmaceutical agents, to the subject in a
pharmaceutically compatible carrier and in an amount effective to
inhibit the development or progression of a disease. Although the
treatment can be used prophylactically in any patient in a
demographic group at significant risk for such diseases, subjects
can also be selected using more specific criteria, such as a
definitive diagnosis of the disease/condition or identification of
one or more factors that increase the likelihood of developing such
disease (e.g., a genetic factor).
[0166] Various delivery systems are known and can be used to
administer chemical compounds and FTIs as therapeutics. Such
systems include, for example, encapsulation in liposomes,
microparticles, microcapsules, recombinant cells capable of
expressing therapeutic molecule(s) (see, e.g., Wu et al., J. Biol.
Chem. 262, 4429, 1987), construction of a therapeutic nucleic acid
as part of a retroviral or other vector, and the like. Methods of
introduction include, but are not limited to, intrathecal,
intradermal, intramuscular, intraperitoneal (ip), intravenous (iv),
subcutaneous, intranasal, epidural, and oral routes. The
therapeutics may be administered by any convenient route,
including, for example, infusion or bolus injection, topical,
absorption through epithelial or mucocutaneous linings (e.g., oral
mucosa, rectal and intestinal mucosa, and the like) ophthalmic,
nasal, and transdermal, and may be administered together with other
biologically active agents. Pulmonary administration can also be
employed (e.g., by an inhaler or nebulizer), for instance using a
formulation containing an aerosolizing agent.
[0167] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment. This may be achieved, for example, and
not by way of limitation, by local infusion or perfusion (e.g.,
during surgery), topical application (e.g., in a dressing),
injection, catheter, suppository, or implant (e.g., implants formed
from porous, non-porous, or gelatinous materials, including
membranes, such as sialastic membranes or fibers), and the like. In
one embodiment, administration can be by direct injection at the
site (or former site) of a tissue that is to be treated. In another
embodiment, the therapeutic are delivered in a vesicle, in
particular liposomes (see, e.g., Langer, Science 249, 1527, 1990;
Treat et al., in Liposomes in the Therapy of Infectious Disease and
Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365,
1989).
[0168] In yet another embodiment, the therapeutic can be delivered
in a controlled release system. In one embodiment, a pump may be
used (see, e.g., Langer Science 249, 1527, 1990; Sefton Crit. Rev.
Biomed. Eng. 14, 201, 1987; Buchwald et al., Surgery 88, 507, 1980;
Saudek et al., N. Engl. J. Med. 321, 574, 1989). In another
embodiment, polymeric materials can be used (see, e.g., Ranger et
al., Macromol. Sci. Rev. Macromol. Chem. 23, 61, 1983; Levy et al.,
Science 228, 190, 1985; During et al., Ann. Neurol. 25, 351, 1989;
Howard et al., J. Neurosurg. 71, 105, 1989). Other controlled
release systems, such as those discussed in the review by Langer
(Science 249, 1527 1990), can also be used.
[0169] The vehicle in which the agent is delivered can include
pharmaceutically acceptable compositions of the compounds, using
methods well known to those with skill in the art. For instance, in
some embodiments, chemical compounds, small molecules, and/or
specifically FTIs typically are contained in a pharmaceutically
acceptable carrier. The term "pharmaceutically acceptable" means
approved by a regulatory agency of the federal or a state
government or listed in the U.S. Pharmacopoeia or other generally
recognized pharmacopoeia for use in animals, and, more
particularly, in humans. The term "carrier" refers to a diluent,
adjuvant, 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, blood plasma medium, aqueous dextrose, and
glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. The medium may also contain
conventional pharmaceutical adjunct materials such as, for example,
pharmaceutically acceptable salts to adjust the osmotic pressure,
lipid carriers such as cyclodextrins, proteins such as serum
albumin, hydrophilic agents such as methyl cellulose, detergents,
buffers, preservatives and the like.
[0170] Examples of 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 therapeutic, if desired, can also
contain minor amounts of wetting or emulsifying agents, or pH
buffering agents. These therapeutics can take the form of
solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations, and the like. The
therapeutic can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, and the like. A more complete
explanation of parenteral pharmaceutical carriers can be found in
Remington: The Science and Practice of Pharmacy (19th Edition,
1995) in chapter 95.
[0171] Embodiments of other pharmaceutical compositions are
prepared with conventional pharmaceutically acceptable
counter-ions, as would be known to those of skill in the art.
[0172] Therapeutic preparations will contain a therapeutically
effective amount of at least one active ingredient, preferably in
purified form, together with a suitable amount of carrier so as to
provide proper administration to the patient. The formulation
should suit the mode of administration.
[0173] The composition of this disclosure can be formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection.
[0174] The ingredients in various embodiments are supplied either
separately or mixed together in unit dosage form, for example, in
solid, semi-solid and liquid dosage forms such as tablets, pills,
powders, liquid solutions, or suspensions, or 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 or saline
can be provided so that the ingredients may be mixed prior to
administration.
[0175] The amount of the therapeutic that will be effective depends
on the nature of the disorder or condition to be treated, as well
as the stage of the disorder or condition. Effective amounts can be
determined by standard clinical techniques. The precise dose to be
employed in the formulation will also depend on the route of
administration, and should be decided according to the judgment of
the health care practitioner and each patient's circumstances. An
example of such a dosage range is 0.1 to 200 mg/kg body weight in
single or divided doses. Another example of a dosage range is 1.0
to 100 mg/kg body weight in single or divided doses.
[0176] The specific dose level and frequency of dosage for any
particular subject may be varied and will depend upon a variety of
factors, including the activity of the specific compound, the
metabolic stability and length of action of that compound, the age,
body weight, general health, sex, diet, mode and time of
administration, rate of excretion, drug combination, and severity
of the condition of the host undergoing therapy.
[0177] The therapeutic compounds and compositions of the present
disclosure can be administered at about the same dose throughout a
treatment period, in an escalating dose regimen, or in a
loading-dose regime (e.g., in which the loading dose is about two
to five times the maintenance dose). In some embodiments, the dose
is varied during the course of a treatment based on the condition
of the subject being treated, the severity of the disease or
condition, the apparent response to the therapy, and/or other
factors as judged by one of ordinary skill in the art. In some
embodiments long-term treatment with the drug is contemplated.
[0178] In some embodiments, sustained localized release of the
pharmaceutical preparation that comprises a therapeutically
effective amount of a therapeutic compound or composition may be
beneficial. Slow-release formulations are known to those of
ordinary skill in the art. By way of example, polymers such as
bis(p-carboxyphenoxy)propane-sebacic-acid or lecithin suspensions
may be used to provide sustained localized release.
[0179] It is specifically contemplated in some embodiments that
delivery is via an injected and/or implanted drug depot, for
instance comprising multi-vesicular liposomes such as in
DEPOFOAM.RTM. drug delivery agent (SkyePharma, Inc, San Diego,
Calif.) (see, for instance, Chamberlain et al., Arch. Neuro.
50:261-264, 1993; Katri et al., J. Pharm. Sci. 87:1341-1346, 1998;
Ye et al., J. Control Release 64:155-166, 2000; and Howell, Cancer
J. 7:219-227, 2001).
IX. Cell-Based Systems for Identifying and Characterizing FTIs for
Treatment
[0180] Also provided herein are in vivo assay systems designed to
show the ability of test compounds (test agents) to inhibit
farnesyltransferase and more specifically, ability of test
compounds to reduce or ameliorate one or more cellular effects
caused by constitutive farnesylation of lamin A.
[0181] If farnesylation is inhibited, maturation of lamin A cannot
be completed and the precursor (prelamin A) accumulates. This
phenomenon has been recognized (see, e.g., Beck et al., J. Cell
Biol. 110:1489-1499, 1990; Kilic et al., J. Biol. Chem.
272:5298-5304, 1997). Thus, inhibition of farnesylation can be
detected by detecting increased accumulation of prelamin A (e.g.,
on a gel based on differential mobility, or using an antibody that
preferentially detects prelamin A). One method for generating a
prelamin A-specific antibody preparation is described in Adjei et
al. (Clin Cancer Res 6:2318-2325, 2000). Detection of prelamin A is
recommended as a marker of farnesyltransferase inhibition in that
same reference. It was shown to be detectable using Western
blotting and immunohistochemical staining, as well as differential
migration on SDS-PAGE.
[0182] Similarly, inhibition of farnesyltransferase can be measured
by detecting changes in processing, and therefore changes in
electrophoretic mobility, of other proteins. By way of example, the
chaperone HDJ-2 [Neckers et al., The Hsp90 chaperone family, In:
D.S. Latchmann (ed.), Stress Proteins, pp. 9-42 (New York:
sprinter-Verlag, 1999)] and the peroxisomal protein Pxf (James et
al., J. Biol. Chem 269:14182-14190, 1994) are good candidate
markers for monitoring farnesyltransferase inhibition (Adjei et
al., Clin Cancer Res 6:2318-2325, 2000).
[0183] In addition, specific examples provided herein describe
constructs useful in cell-based systems. These systems can be used,
for instance, in studying the effectiveness of specific FTIs (or
other candidate therapeutic compounds) to ameliorate symptoms
(particularly, cellular defects) of constitutive farnesylation of
lamin protein forms. Specific systems therefore exploit methods of
detecting and analyzing mis-localization of a farnesylated lamin,
mis-localization of a non-farnesylated lamin, nuclear membrane
disruption, aggregation of lamin protein, nuclear lobulations,
nuclear blebbing, cytoskeleton disruption, early senescence,
apoptosis (e.g., through measurement of Annexin V), reduced
secretion of MMP-3, and so forth. It is contemplated that certain
of such cell-based systems could be used in a high-throughput
format, in order to more rapidly analyze the effect and
effectiveness of known and putative inhibitory molecules.
[0184] Examples of described constructs, cell-based systems, and
transgenic animals are specifically contemplated for use in
characterizing, testing, selecting, and identifying compounds that
may be putative farnesyltransferase inhibitory.
[0185] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the invention to the particular features or
embodiments described.
EXAMPLES
Example 1
Incomplete Lamin a Processing in HGPS Leads to Nuclear
Abnormalities
[0186] This example describes methods for detecting lamin A in
cells, and examining cellular characteristics that result from
expressing progerin.
[0187] GFP-tagged normal and mutant LMNA expression constructs have
been created, and normal and HGPS fibroblasts have been transfected
with these constructs in order to examine protein localization and
effect on nuclear structure. Expression of the mutant lamin A
protein (progerin) in normal fibroblasts caused abnormal nuclear
morphology including mislocalization of progerin to foci at the
nuclear periphery and nuclear blebbing similar to that seen in HGPS
fibroblasts. Expression of normal lamin A in HGPS fibroblasts did
not significantly correct the nuclear blebbing phenotype.
Furthermore, the normal GFP-lamin A was mislocalized, indicating
that the presence of the endogenous mutant can act in a
dominant-negative fashion. The progerin deletion could have a
number of consequences which could give rise to this abnormal
cellular phenotype, including abnormal protein processing and
deletion of critical protein binding domains. The deleted region
includes a protein cleavage site that normally removes 15 amino
acids including a CAAX box (SEQ ID NO: 31) farnesylation site.
Methods
[0188] Constructs:
[0189] Normal and mutant alleles of lamin A/C were cloned by
isolating RNA from the cell lines AG11513a and NF, respectively. An
oligo dT primer was used for first strand synthesis using the
SUPERSCRIPT.RTM. reverse transcriptase kit (Invitrogen). The first
strand cDNA was used as a template to amplify normal and mutant
products using the Platinum Taq high fidelity kit from Invitrogen
with the primers laminAF1 (SEQ ID NO: 1) and laminAR1 (SEQ ID NO:
2). Due to the GC-rich nature of the template, DMSO and betaine
were added to final concentrations of 4% and 500 mM, respectively.
The reaction was run for 20 cycles with an annealing temperature of
62.degree. C. and the resulting product was used for TOPO TA
cloning (Invitrogen). These cDNA constructs were verified by
sequencing and used as templates for subcloning into the pEGFP-C2
and pRevTRE vectors (Clontech).
[0190] Cell Culture:
[0191] Cell lines used were normal fibroblast lines, GM08398 and
HGMDFN090, and HGPS fibroblast lines: P01 (HGADFN003), HGADFN004,
HGADFN136, AG03513d, AG10750 and AG11498. Fibroblasts were cultured
in minimal essential medium (Gibco, #10370-021) supplemented with
15% FBS (HiClone), 2 mM L-glutamine, penicillin (50 U/ml) and
streptomycin (50 mg/ml). The Amphotropic Phoenix retroviral
packaging line (Peter Nolan, Stanford University) was maintained in
DMEM supplemented with 10% FBS, 2 mM L-glutamine, penicillin (50
U/ml) and streptomycin (50 mg/ml).
[0192] Retroviral Constructs:
[0193] The pRevTetOff vector and pRevTRE constructs were
transfected using FUGENE.RTM.6 transfection reagent into the
Amphotropic Phoenix retroviral packaging line (Peter Nolan,
Stanford University). Forty-eight hours after transfection the
media containing retrovirus was filtered through a 0.22 micron
filter and used immediately or stored at -70.degree. C. Retroviral
infection was performed by exposing target cells to a 1:1 mix of
retroviral supernatant and growth medium with 4 .mu.g/ml
polybrene.
[0194] GFP Localization:
[0195] Normal and HGPS fibroblasts were plated on coverslips in six
well plates or in chambered microscope slides (BD Biosciences) 24
hours prior to transfection. Fibroblasts were transiently
transfected with the GFP-lamin A or GFP-progerin expression
constructs using FUGENE.RTM.6 transfection reagent (Roche).
Seventy-two hours later the cells were washed two times with PBS
(pH 7.2) and fixed for 20 minutes at 4.degree. C. with 4%
paraformaldehyde in PBS containing 62.5 .mu.g/ml LPC palmitoyl and
1 U/ml rhodamine phalloidin (Molecular Probes) to stain the
cytoskeleton. Following three washes with PBS, Prolong Gold
mounting medium with DAPI (Molecular Probes) was applied to the
coverslips which were then inverted and placed on microscope
slides.
[0196] Immunocytochemistry:
[0197] Immunocytochemistry (ICC) was performed as previously
described. Cells were cultured on coverslips in 6-well plates for
72 hours. Coverslips were washed twice with PBS and fixed with 4%
paraformaldehyde in PBS with 0.18% Triton X-100 for 20 min at
4.degree. C. Coverslips were washed 3 times with PBS and incubated
30 min at 37.degree. C. in blocking buffer (1% BSA in PBS). The
cells were probed with 1:10 dilution of the lamin A specific
antibody (USBiological) in blocking buffer for 1 hour at room
temperature followed by washing three times with PBS. Secondary
antibody (Goat Anti-Mouse IgG, FITC) was diluted 1:1000 in blocking
buffer and applied to the coverslips for 1 hour at room
temperature. After washing three times for 5 minutes with PBS, the
coverslips were inverted and placed on slides with ProLong Gold
mounting medium containing DAPI (Molecular Probes).
[0198] Immunoprecipitation:
[0199] Cells from nearly confluent T75 flasks were harvested and
lysed in RIPA buffer containing a protease inhibitor cocktail
(Roche). Cell pellets were vortexed and sonicated briefly to ensure
thorough lysing. 500 mg of total cellular protein in a final volume
of 500 .mu.l from each sample was immunoprecipitated as follows.
Cell lysate was precleared by incubating for 1 hour with protein G
sepharose at 4.degree. C. on a nutator mixer. The protein G
sepharose was removed by a brief spin and the supernatant was
incubated with 100 .mu.l anti-lamin A/C antibody (Abcam ab8984)
overnight at 4.degree. C. Fifty .mu.l protein G Sepharose was added
to each sample and incubated for 3 hour at 4.degree. C. After
washing four times with RIPA buffer, 50 .mu.l 2.times. NuPAGE
sample buffer with reducing agent (Invitrogen) was added to the
protein G pellet, heated to 100.degree. C. for 10 min, and loaded
onto a 4-12% Bis-Tris NuPAGE gel (Invitrogen).
[0200] Immunoblotting:
[0201] Proteins were transferred to Immobilon-P membrane
(Millipore) using a Trans-Blot semi-dry electrophoretic transfer
cell (BioRad) blocked with 5% dry milk in TBST and probed with
lamin A/C specific antibodies according to standard protocols.
Results
GFP Localization
[0202] When GFP-tagged normal lamin A was expressed in normal
fibroblasts, the nuclei had an oval shape with fluorescence evenly
distributed at the periphery of the nucleus (FIGS. 4A and 4B).
Expression of the GFP-tagged progerin in normal fibroblasts caused
abnormal nuclear morphology, similar to that seen in HGPS
fibroblasts including nuclear blebbing, and the mislocalization of
the GFP-tagged progerin into nuclear foci in many cells (FIG. 4C).
Expression of normal lamin A in HGPS fibroblasts for 72 hours did
not significantly correct the nuclear blebbing phenotype seen in
these cells, and the normal GFP-lamin A was mislocalized,
indicating that the presence of the endogenous mutant can cause
mislocalization of the normal GFP-tagged lamin A. These experiments
were repeated using confocal microscopy in order to verify these
results and gain very high resolution images that better show the
localization of GFP-progerin (FIG. 6). The most commonly seen
localization pattern for the GFP-progerin 72 hours
post-transfection appears as thick regions in the nuclear lamina.
These were also detected by ICC in HGPS fibroblasts with less
resolution most likely due to a lack of availability of the epitope
within these aggregates (FIG. 5).
[0203] GFP-lamin A and GFP-progerin were also expressed from
TET-OFF.RTM. retroviral expression vector constructs that allow for
sustained expression and modulation of expression to address the
possibility of results due to over-expression of the fusion
proteins. Reducing expression of GFP-progerin (as measured by
Western blotting) did not significantly alter the localization of
the mutant protein into aggregates at the nuclear periphery.
Progerin Processing
[0204] In order to determine if the progerin protein retains a
farnesyl group an immunoprecipitation (IP) was performed using anti
lamin A/C antibodies (Abcam ab8984). The IP step was necessary
because as many as 0.1% of cell proteins are modified by the
addition of farnesyl or geranylgeranyl group. Progerin was
efficiently detected following IP as a protein of approximately 74
kDa that runs between lamin C (70 kDa) and lamin A (78.5 kDa) on an
immunoblot, consistent with the loss of 50 amino acids from lamin A
(FIG. 7C). The presence of a .sup.3H-labeled farnesyl group on the
progerin protein was detected by fluorography of the
immunoprecipitated proteins. A farnesyl group was not detected on
normal lamin A so the normal protein was completely processed in
these cells. The addition of 2 .mu.M FTI prevented the
farnesylation of progerin and lamin A. In addition, the pre-lamin A
band in these lanes was shown by the presence of a larger band
since farnesylation was necessary for the subsequent processing
steps including removal of the C-terminal 18 amino acids.
Discussion
[0205] To date, there have been seven diseases associated with
mutations in the LMNA gene. The disorders include muscular
dystrophy, lipodystrophy and neuropathy, and the tissues most
affected in these disorders are striated muscle, adipose tissue and
bone (reviewed in Burke & Stewart, Nat Rev Mol Cell Biol
3:575-585, 2002). The remarkable heterogeneity and phenotypic
differences of the laminopathies are reflected in the distribution
of mutations in LMNA. Virtually all HGPS patients have the same
mutation creating an abnormal splice donor site in exon 11 of the
LMNA gene. The mis-splicing creates a protein missing 50 amino
acids in a globular domain near the C-terminus. Mutations causing
Dreifuss muscular dystrophy (EDMD2) are distributed throughout the
gene, while those resulting in familial dilated cardiomyopathy type
1 (CMD1A) are mainly clustered within the coiled-coil domain,
suggesting that the latter may act by disrupting dimerization or
subsequent polymerization of lamin A/C. In contrast, the mutations
found in individuals with HGPS, Dunnigan familial partial
lipodystrophy (FPLD), and mandibuloacral dysplasia (MADA or MAD)
are found within exons encoding the C-terminal globular domain of
lamin A/C and, more specifically, are restricted to just a few
codons in exons 8, 9 and 11 (Eriksson et al., Nature 423:293-298,
2003; Shackleton et al., Nat Genet 24:153-156, 2000; Speckman et
al., Am J Hum Genet 66:1192-1198, 2000; Novelli et al., Am J Hum
Genet 71:426-431, 2002). Abnormal nuclear morphology is seen in
many of the laminopathies, including Emory (EDMD2) and FPLD, so it
seems unlikely that this cellular phenotype alone accounts for the
HGPS-specific disease phenotypes.
[0206] We and others have described the mis-splicing caused by the
activation of a cryptic splice site due to the codon 608 mutation
in HGPS. The mis-splicing results in loss of 150 nucleotides
between the cryptic splice site and the end of exon 11. The frame
of the transcript is retained so that the final nine codons from
exon 12 are transcribed including a CAAX box (SEQ ID NO: 31)
farnesylation site. However, the deletion of 50 amino acids may
have numerous consequences, including deletion of protein binding
domains and deletion of the cleavage site necessary for normal
protein processing and predicted removal of the farnesyl group from
the mature protein. Incomplete processing of lamin A appears to be
an important factor as mutations in ZMPSTE24 (the protease that
performs the final cleavage step removing the farnesyl group) are
found in patients with a severe form of mandibuloacral dysplasia.
Although the conserved cleavage site is missing in progerin, it
remained possible that the protein was still cleaved as there are
potential sites in the progerin protein that may have substituted
for the missing cleavage sequence. Thus, while maintenance of the
farnesyl group is predicted, it had not been formally proven.
[0207] Data presented in this example show that the progerin
protein remains farnesylated and is not cleaved. The retention of
the farnesyl group may have numerous consequences. Farnesyl groups
increase lipophilicity and are involved in membrane association.
Also, farnesyl groups are involved in protein interactions. In
fact, the protein Narf has been shown to interact only with the
farnesylated form of lamin A (Barton & Worman, J Biol Chem.
274:30008-30018, 1999). Retention of the farnesyl group on the
progerin protein and on the cleavage minus mutant alters the
localization of these proteins in comparison to the normal lamin A.
This abnormal distribution of the nuclear lamin A may contribute to
the loss of integrity of the lamina and thus result in the abnormal
nuclear phenotype observed in HGPS cells and when these proteins
are expressed in normal cells.
[0208] N-terminal GFP-lamin A fusions have been shown previously to
display localization patterns consistent with localization of the
non-tagged protein. The fusion protein has the added advantage of
showing lamin structures that are difficult to detect using ICC
because epitopes are not available.
Example 2
Cleavage Mutant of Lamin A
[0209] As shown in Example 1, the C-terminus of progerin was not
cleaved and the protein remained farnesylated. This example
provides the construction of a cleavage-site mutation in lamin A,
and compares its processing and cellular effects with those of
progerin. Transfection of normal cells with the codon 647 cleavage
site mutation construct was found to give rise to similar nuclear
abnormalities as those seen in progeria cells. These results
indicate that abnormal farnesylation of progerin plays a major role
in the abnormal cellular phenotype.
Methods
Creation of Cleavage Minus Mutant
[0210] The L647R mutant was introduced into the pRevTRE-GFP-lamin A
construct using a PCR-based site directed mutagenesis. Two
overlapping PCR products were generated using the following primer
pairs: PCR product 1 using the primers LMNA-1320-For (SEQ ID NO: 3)
and LMNA-L647R-Rev (SEQ ID NO: 4), and PCR product 2 using the
primers LMNA-L647R-For (SEQ ID NO: 5) and pRevTRE-3700-Rev (SEQ ID
NO: 6). The reverse primer for product 1 is the complement sequence
to the forward primer for product 2 and both of these primers
contain a single mismatch with the target sequence that will
generate the L647R mutation.
[0211] Both products were gel purified and used together as the
template for a third PCR reaction using the primers LMNA-1390-For
(SEQ ID NO: 7) and pRevTRE-3440-Rev (SEQ ID NO: 8). Although the
template is two overlapping fragments, a single PCR product
results, which was gel purified and cloned using the TOPO.RTM. TA
cloning kit (Invitrogen). The resulting insert was subcloned into
the pRevTRE-GFP-lamin A construct using Mfe I and Cla I restriction
endonucleases. The resulting construct, pRevTRE-GFP-LA(L647R) was
sequenced to verify the presence of the introduced mutation without
any random base changes due to the use of PCR to generate the
construct.
Results and Discussion
[0212] A mutant lamin A was engineered to create a L647R amino acid
change in the cleavage site of lamin A, so that the effect of
retaining the farnesyl group could be assessed without the 50 amino
acid deletion that occurs in the progerin protein. The L647R
mutation changed a conserved base in the cleavage site of lamin A
so that ZMPSTE24 could not cleave the protein, leaving it
farnesylated.
[0213] A GFP-tagged cleavage-minus mutant lamin A (L647R) displayed
a localization pattern nearly identical to the localization of
GFP-tagged progerin when expressed in normal fibroblasts (FIG. 8).
Its expression caused a significant increase in abnormal nuclear
morphology, similar to the effect of the GFP-progerin construct
(Example 1) (FIG. 9). This result demonstrates that incomplete
processing of lamin A such that it retains the farnesyl group can
cause morphological changes in cells similar to those caused by
progerin expression.
Example 3
Farnesyltransferase Inhibitor Affects HGPS Nuclear
Abnormalities
[0214] This example demonstrates that treatment of cells expressing
progerin with a farnesyltransferase inhibitor reverses cellular
morphology defects. The FTI used in this example, PD169451, was a
gift from Pfizer.
Effect of FTI, PD169451, on Transiently Expressed GFP-Lamin A and
GFP-Progerin
[0215] Normal and HGPS fibroblasts were plated on coverslips in 6
well plates 24 hours prior to transfection. Normal fibroblasts were
transiently transfected with the GFP-lamin A or GFP-progerin
expression constructs using FUGENE.RTM.6 transfection reagent
(Roche). The following day, the medium was replaced with growth
medium containing 0 nM, 100 nM, 500 nM or 1 mM of the FTI,
PD169451. Forty-eight hours later the cells were washed two times
with PBS (pH 7.2) and fixed for 20 min at 4.degree. C. with 4%
paraformaldehyde in PBS containing 62.5 .mu.g/ml LPC palmitoyl and
1 U/ml rhodamine phalloidin (Molecular Probes) to stain the
cytoskeleton. Following three washes with PBS, 10 .mu.l of ProLong
Gold mounting medium with DAPI (Molecular Probes) was applied and
the coverslips, which were inverted onto a glass microscope
slide.
Effect of FTI, PD169451, on the Nuclear Morphology of Normal and
HGPS Fibroblasts
[0216] Normal and HGPS fibroblasts were maintained in T75 tissue
culture flasks with 0 nM, 10 nM, and 100 nM concentrations of the
FTI, PD169451, added to the growth medium. The medium was changed
every 2 days and the cells were split when they were approximately
80% confluent. Three days prior to each time point, a flask was
trypsinized and cells were placed in chambered microscope slides
(BD Biosciences) and maintained in the appropriate FTI
concentration. Seventy-two hours later the cells were washed two
times with PBS (pH 7.2) and fixed for 20 minutes at 4.degree. C.
with 4% paraformaldehyde in PBS containing 62.5 .mu.g/ml LPC
palmitoyl and 1 U/ml rhodamine phalloidin (Molecular Probes) to
stain the cytoskeleton. Following three washes with PBS, 10 .mu.l
of ProLong Gold mounting medium with DAPI (Molecular Probes) was
applied and the cells were overlaid with a coverslip.
Results and Discussion
[0217] The effects of several concentrations of the specific
farnesyltransferase inhibitor Pfizer PD 169451 on nuclear
morphology and localization of lamin A and progerin were examined.
Exposure of 100 nM FTI for 72 hours had a significant effect on the
localization of transiently expressed GFP-lamin A and GFP-progerin
(FIGS. 7A and 7B respectively). Lamin A and progerin were localized
in very similar fashion to intranuclear filaments when exposed to
the FTI rather than localizing to the nuclear periphery (compare
FIGS. 7A and 7B with FIGS. 6A and 6B). In addition, short-term
exposure to the FTI had a significant effect on the nuclear
morphology of fibroblasts expressing GFP-progerin. These
fibroblasts had a 33% decrease in the percentage of cells with
abnormal nuclear morphology (FIG. 7D). To verify this result in
HGPS fibroblasts, the AG11498 line was exposed to various FTI
concentrations for three days. Treating these HGPS cells resulted
in as much as a 40% drop in the percent of cells with abnormal
nuclear morphology (FIG. 7E).
[0218] A longer duration analysis on the effects of FTI on the
nuclear morphology of normal and HGPS fibroblasts revealed that a
sustained low dose of 10 nM substantially reduced the percentage of
cells displaying abnormal nuclear morphology (FIG. 10). High doses
of the FTI, greater than 200 nM, caused cells to stop dividing
after a week to 10 days, and caused an increase in abnormal nuclear
morphology in normal fibroblasts. However, 10 nM FTI did not
substantially alter the nuclear morphology of normal cells and
significantly lowered the percentage of abnormal nuclei in HGPS
fibroblasts and in normal fibroblasts expressing GFP-progerin. Four
HGPS cell lines were maintained in growth media containing 10 nM or
100 nM FTI. After two days exposure to 10 nM FTI the four HGPS cell
lines demonstrated approximately a 40% reduction in the percent of
cells with abnormal nuclei. At six days, the HGPS lines displayed a
72%, 70%, 45%, and 27% reduction in abnormal nuclei after six days
of 10 nM FTI. After 14 days, three HGPS lines each had at least a
70% decrease in abnormal nuclei and one HGPS cell line (HGADFN004)
had a 12% drop in abnormal morphology. This cell line failed to
grow at the 14 day time point when exposed to 100 nM FTI. Exposure
to the FTI causes some abnormal nuclear morphology in normal
fibroblasts in a dose dependent manner.
[0219] Because the progerin protein maintains the farnesyl group,
FTIs might improve the nuclear morphology of HGPS fibroblasts and
normal fibroblasts expressing progerin by sequestering the mutant
protein into the intranuclear filaments. These results showed a
significant reduction in the abnormal nuclear phenotype in cells
treated with an FTI. The FTI used in these experiments, PD169451,
is an isoprene competitor which is specific for farnesyltransferase
and does not inhibit the related geranylgeranyl transferase I. FTIs
have been investigated as anticancer agents because farnesylation
of Ras is necessary for Ras signaling and biological activity.
Other FTIs have been used in clinical studies and are well
tolerated by patients. The improvement in the morphology of the
HGPS cells when exposed to low doses of the FTI indicates that
these agents may be useful in treating the disease. The effect of
these drugs in animal models of HGPS may be analyzed to determine
if there is improvement in the overall phenotype of the animals.
The adverse effects of higher concentrations of the FTI on nuclear
morphology and cell growth and the reduced effect on a higher
passage HGPS line may indicate that there is a therapeutic window
for effective concentrations of the drug and time of treatment for
the patient.
Example 4
Effects of FTI Treatment on Expression of L647R Cleavage Mutant
[0220] The effects of exposure to the FTI PD169451 on cells
expressing the lamin A L647R cleavage mutant are being tested.
Based on the results observed with progerin (Example 3), it is
believed that the FTI will cause the GFP-tagged L647R mutant to
localize to intranuclear filaments, as observed when GFP-lamin A
and GFP-progerin are expressed in cells exposed to the FTI. This
result is expected because lamin A (and its mutant forms) must be
farnesylated in order to be localized to the nuclear lamina. The
FTI causes these forms of lamin A to accumulate in the interior of
the nucleus. It is also predicted that the increase in abnormal
nuclear morphology observed when the GFP-L647R mutant is expressed
in normal fibroblasts will be alleviated by FTI treatment, as
observed herein for normal fibroblasts expressing GFP-progerin when
treated with the FTI.
[0221] By way of example, GFP-lamin A and GFP-progerin were
expressed from TET-OFF.RTM. retroviral expression vector constructs
that allowed for sustained expression and modulation of expression
to address the possibility of results due to over-expression of the
fusion proteins when the proteins were transiently expressed. In
addition, a cleavage minus mutant, GFP-LA(L647R), was included to
examine the effect of retaining the farnesyl group without the 50
amino acid deletion found in progerin.
[0222] When no tetracycline was present, the GFP fusions were
expressed at relatively high levels as measured by Western blot
(FIG. 11 D).
[0223] Addition of 0.05 mg/ml tetracycline allowed for reduced
expression of the GFP fusions such that near-endogenous levels were
obtained, while 8 mg/ml tetracycline reduced, but did not
altogether prevent, expression from these constructs (FIG. 11 D).
When GFP-progerin was stably expressed in normal fibroblasts (0.05
mg/ml tetracycline) at levels similar to progerin levels in HGPS
fibroblasts, 10 nM FTI substantially reduced the percent abnormal
nuclei by 71% (p<0.0001) (FIG. 11B).
[0224] When GFP-tagged L647R mutant was expressed at intermediate
levels (0.05 mg/ml tetracycline), the increase in abnormal nuclear
morphology was reduced 67% by exposure to 10 nM FTI (p<0.0001)
(FIG. 11 B). The improvement in abnormal nuclear morphology with
intermediate expression levels of GFP-progerin and GFP-LA(L647R)
was actually less profound at the higher concentrations of the FTI
(100 nM) in comparison to the decrease observed for 10 nM (FIG. 11
B). Treatment with 10 nM FTI could not overcome the effects of very
high expression levels of the GFP-progerin or GFP-LA(L647R) that
were obtained when no tetracycline was present to repress
expression (FIG. 11 A). However, the cleavage minus mutant may be
more tractable to FTI treatment at the highest expression level,
especially when exposed to 100 nM FTI, perhaps due to slightly
lower expression levels. Some leaky expression was observed even at
8 mg/ml tetracycline, and these low levels did affect nuclear
morphology, causing a moderate increase in the percent abnormal
nuclei in the GFP-progerin and GFP-LA(L647R) cells (FIG. 11 C).
Thus, it is unlikely that the effects seen with the progerin
constructs are due solely to protein over expression.
[0225] As with transient expression, stable expression of GFP-lamin
A did not have a significant effect on nuclear morphology, and the
GFP signal was evenly distributed at the nuclear periphery (FIG. 12
A).
[0226] When GFP-progerin was expressed in normal fibroblasts at
levels near what is observed in HGPS patients, nuclear aggregates
at the periphery of the nucleus and intranuclear structures were
observed, along with a substantial increase in abnormal nuclear
morphology including folding and blebbing of the nuclear membrane
(FIG. 12 D). In addition, chromatin staining by DAPI was altered
with a loss of staining often observed at the nuclear periphery and
in regions adjacent to the progerin aggregates. Expression of
intermediate levels of the cleavage minus mutant, GFP-LA(L647R),
had very similar effects on localization of the GFP signal and on
nuclear morphology as the GFP-progerin (FIG. 12 G). Also, as
observed in the transient expression studies, the FTI caused the
GFP-lamin A and GFP-progerin to localize to intranuclear filaments
when expressed in cells exposed to the FTI at 100 nM
concentrations. Localization of GFP-tagged L647R mutant was also
redistributed to intranuclear filaments when exposed to 100 nM FTI
(FIG. 12 I). However, 10 nM FTI did not lead to this drastic
reorganization of GFP signal for any of the fusion proteins.
Rather, alterations in GFP localization at this FTI concentration
were less noticeable (FIGS. 12 B, E and H).
[0227] Material included in this example was also published as
Glynn & Glover, Hum Mol Genet 14: 2959-2569, 2005, Sep. 6,
2005, which is incorporated herein by reference in its
entirety.
Example 5
Testing FTIs in HGPS, Experimental Protocols
[0228] This example describes production of a series of expression
constructs for lamin A, progerin, and derivative proteins having
mutations in the CAAX box (SEQ ID NO: 31). Methods are provided for
analyzing such expression constructs in vivo. These methods are
examples of analyses which can be used for determining the
effectiveness of farnesyltransferase inhibitors (FTIs), potential
FTIs, and other drugs and compounds, for reversing or reducing
cellular defects caused by accumulation of progerin or other
farnesylated proteins.
Mutant Construction
[0229] A set of lamin A CAAX (SEQ ID NO: 31) mutants was generated,
including CSIM (SEQ ID NO: 32) to CSIL (SEQ ID NO: 34) and to SSIM
(SEQ ID NO: 33) mutants in constructs expressing wildtype or del150
lamin A proteins (Map 1 of FIG. 11). These expression constructs
were made according to protocol using the QUIKCHANGE.RTM.II XL
Site-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.).
Primers were designed using QUIKCHANGE.RTM. site directed
mutagenesis Primer Design (Stratagene), synthesized and purified by
polyacrylamide gel electrophoresis at Integrated DNA Technologies,
Inc. (Coralville, Iowa).
[0230] Primer sequences for the cDNA constructs were as follows:
SSIM_Sense (SEQ ID NO: 11); SSIM_Anti-sense (SEQ ID NO: 12);
CSIL_Sense (SEQ ID NO: 13); and CSIL_Anti-sense (SEQ ID NO: 14).
Primer sequences for the minigene constructs were as follows:
SSIM_Sense (SEQ ID NO: 15); SSIM_Anti-sense (SEQ ID NO: 16);
CSIL_Sense (SEQ ID NO: 17); and CSIL_Anti-sense (SEQ ID NO:
18).
[0231] The "del150" lamin A framework provides a protein that has
deleted from it the same 50-amino acids that are deleted in the
HGPS protein. Map 1 of FIG. 13 provides an overview of these
constructs. Specific examples of entire coding sequence (SEQ ID
NOs: 19, 21, 23, 25, 27, and 29) for specific lamin A proteins (SEQ
ID NOs: 20, 22, 24, 26, 28, and 30) are shown herein, which
correspond respectively to constructs pEGFP_LA_myc_wildtype CSIM
(the "normal" lamin A), pEGFP_LA_myc_wildtype CSIL,
pEGFP_LA_myc_wildtype SSIM, pEGFP_LA_myc_del150 CSIM,
pEGFP_LA_myc_del150 CSIL, and pEGFP_LA_myc_del150 SSIM.
[0232] Cell Culture:
[0233] For immunofluorescence, HeLa cells were plated in chamber
slides at 25,000 cells per chamber and cultured in DMEM containing
10% FBS, penicillin-streptomycin, and L-glutamine until 90-95%
confluent prior to transfection.
[0234] Transfection:
[0235] The following constructs were transfected by lipofection
into the HeLa cells using the LIPOFECTAMINE.RTM. 2000 transfection
reagent (Invitrogen, Carlsbad, Calif.) transfection procedure for
DNA:
[0236] pEGFP_LA_myc_del150 CSIL cDNA;
[0237] pEGFP_LA_myc_del150 SSIM cDNA;
[0238] pEGFP_LA_myc_wildtype CSIL cDNA;
[0239] pEGFP_LA_myc_wildtype SSIM cDNA;
[0240] pEGFP_LA_myc_wildtype CSIM cDNA (-control);
[0241] pEGFP_LA_myc_del150 CSIM cDNA (+control); and
[0242] mock transfection vector only (CMV driven EGFP vector with
no insert).
[0243] Optionally, for each transfection, Western analysis can be
performed to determine levels of progerin, prelamin, and lamin A.
Using the myc-epitope tag engineered into these constructs,
endogenous protein can be differentiated from the protein produced
from the transfected construct.
[0244] Transfection efficiency was determined by the visual
inspection of EGFP signal versus the DAPI staining of nuclei.
Transfection efficiency for cells on all slides was estimated to be
30-40%.
[0245] Fluorescence and Electron Microscopy:
[0246] At different time points (e.g., 48 and 72 hours after
transfection), cells grown on coverslips were washed with PBS and
fixed with 3.7% formaldehyde in PBS for 10 minutes at room
temperature. After fixation, cells were washed three times with
PBS, then permeabilized with 0.5% Triton X-100 in PBS for 5 minutes
at room temperature, then rinsed with PBS. Cells were then overlaid
with primary antibody, a rabbit anti-prelamin A from Dr. Michael
Sinensky (1:5000 in PBS with 10% FBS; Cancer Research 54:
3229-3232; 1994), and incubated at room temperature for 1-2 hours.
After removal of the primary antibodies, samples were washed once
with PBS and incubated for 30 minutes in the dark at room
temperature with a mixture of affinity-purified
rhodamine-conjugated goat anti-rabbit IgG (2 .mu.g/ml; Alexa fluor
594(red); Molecular Probes, Eugene, Oreg.), washed three times with
PBS, and dehydrated in increasing concentrations of ethanol baths
(70, 90, 100%) for two minutes per solution. 20 .mu.l of a 50:50
Vectashield:Vectashield with DAPI (Vector Laboratories, Burlingame,
Calif.) mixture was placed on the center of each pad and a
coverslip was placed on top. Slides were then examined on a Zeiss
Axioplan fluorescence microscope. LMNA was viewed using natural
EGFP expression and prelamin A with the Sinensky Ab and Alexa
Fluor594.
Results and Discussion
[0247] After 48 hours of incubation, the HeLa cells transfected
with the pEGFP_LA_myc_wildtype CSIM cDNA construct (the negative
control) appeared normal with the nuclear rim of transfected cells
expressing the green EGFP marker for lamin A. These nuclei appeared
to have normal ovoid and elliptical morphologies by both the DAPI
and EGFP staining. In contrast, the positive control (the
pEGFP_LA_myc_del150 CSIM cDNA progerin construct) displayed
dramatic nuclear blebbing and lobulations by both DAPI and EGFP
staining, identical to the blebbing seen in the nuclei of patients
with HGPS.
[0248] The mutant pEGFP_LA_myc_wildtype SSIM cDNA construct
demonstrated normal nuclear morphology, as would be expected.
However, in this case, all of the EGFP staining appeared as
aggregates within the nucleoplasm, with no EGFP at the nuclear rim,
representing the new localization of the lamin A when it is unable
to be farnesylated and directed to the nuclear membrane. Similarly,
these nucleoplasmic aggregates were seen in the mutant
pEGFP_LA_myc_del150 SSIM cDNA progerin construct. In this case
however, there was no nuclear blebbing or lobulations as would be
expected with the progerin construct.
[0249] The pEGFP_LA_myc_wildtype CSIL cDNA construct provided more
of an intermediate picture. All the cells maintained their ovoid,
elliptical shape in terms of morphology. Lamin A localization as
determined by EGFP expression was a mix between many cells with
normal nuclear membrane staining and many others with staining
occurring only in the nucleoplasmic aggregates as seen in the SSIM
mutants.
[0250] Likewise, the pEGFP_LA_myc_del150 CSIL cDNA construct also
displayed a mixed phenotype, with a moderate amount of blebbing
occurring in some cells mixed with many completely normal looking
cells. Similarly, approximately half of the cells appeared to have
normal lamin A EGFP staining at the nuclear rim, while the other
half only expressed lamin A in nucleoplasmic aggregates.
[0251] After incubating transfected cells for 72 hours, all of the
above results were unchanged except those concerning the CSIL
mutants. In the case of the pEGFP_LA_myc_wildtype CSIL cDNA
construct, there was a much greater percentage of cells appearing
to express lamin A only in the nucleoplasm, and far fewer cells
appearing like normal wildtype cells with lamin A only at the
nuclear rim. The pEGFP_LA_myc_del150 CSIL cDNA progerin construct
also appeared more like the SSIM del150 mutant, with far fewer
blebbed cells and greater percentage of cells with normal ovoid,
elliptical shapes and lamin A EGFP expression localized only in the
nucleoplasm.
Example 6
Minigene Constructs for Lamin A Mutants
[0252] Additional constructs can be made, in which the intron
between the LMNA exon 11 and 12 is included. A progerin protein can
be produced from such constructs if a mutation that activates the
cryptic splice site found in HGPS is included. These constructs are
referred to as "minigene" constructs. An overview of a
representative minigene construct is shown in Map 2 of FIG. 13.
[0253] The following constructs are made and used to transfect
cells as described in Example 5:
[0254] pEGFP_LA.sub.--75_myc_del150 CSIL minigene;
[0255] pEGFP_LA.sub.--75_myc_del150 SSIM minigene;
[0256] pEGFP_LA.sub.--77_myc_wt CSIL minigene;
[0257] pEGFP_LA.sub.--77_myc_wt SSIM minigene;
[0258] pEGFP_LA_myc_wt CSIM minigene (-control);
[0259] pEGFP_LA_myc_del150 CSIM minigene (+control); and
[0260] mock transfection vector only (CMV driven EGFP vector with
no insert).
[0261] Cells transfected with minigene constructs are subjected to
analysis as described in Example 5. These minigene constructs will
be useful to determine if the efficiency of splicing may affect
results such as those noted in Example 5.
[0262] Also contemplated are minigene constructs in which the
expression of the lamin A/progerin/mutation protein is under
inducible control. By way of example, tetracycline inducible
constructs are contemplated (see, for instance, Map 3 of FIG.
13).
Example 7
Treatment with FTIs
[0263] Constructs such as those described in Examples 5 and 6 can
be used in cell-based and animal expression systems to further
characterize known FTIs and potential farnesyltransferase
inhibitory compounds, in particular as regards the effectiveness of
such compounds to ameliorate cellular defects associated with
constitutive farnesylation of lamin A. By way of example, FTIs are
reconstituted in DMSO and stored at -20.degree. C. until used. The
following concentrations of two specific FTIs will be used to
determine their ability to prevent or reduce nuclear blebbing in
the pEGFP_myc_del150 (CSIM) progerin construct and pEGFP_myc_wt
(CSIM) LMNA construct:
TABLE-US-00001 R115777 SCH66336 50 nM (.05 .mu.M) 50 nM (.05 .mu.M)
100 nM (.1 .mu.M) 100 nM (.1 .mu.M) 200 nM (.2 .mu.M) 200 nM (.2
.mu.M) 500 nM (.5 .mu.M) 500 nM (.5 .mu.M) 1000 nM (1 .mu.M) 1000
nM (1 .mu.M) 2000 nM (2 .mu.M) 2000 nM (2 .mu.M) 5000 nM (5 .mu.M)
5000 nM (5 .mu.M) 10000 nM (10 .mu.M) 10000 nM (10 .mu.M)
[0264] Cells are cultured in media containing one of the indicated
levels of FTI for 4-5 days or until 90-95% confluent prior to
transfection. Transfected cell samples are examined by Western blot
analysis to determine endogenous and transfected levels of
progerin, prelamin, and lamin A.
Morphometric Analysis
[0265] To determine the ability of FTIs to prevent nuclear
blebbing, 200 nuclei from both treated and untreated cell cultures
transfected with the pEGFP_myc_del150 (CSIM) progerin construct or
the pEGFP_myc_wt (CSIM) LMNA construct are scored either as
lobulated, if they contain more than two lobulations, or not
lobulated. These determinations are made by using a double-blind
approach, followed by averaging the two data sets. Degree of
farnesylation inhibition is determined by the percentage of cells
demonstrating the presence of prelamin A.
[0266] Contour analysis as in Goldman et al. (Proc. Natl. Acad.
Sci. U.S.A. 8963-8968, 2004) can be used to accurately quantitate
nuclear morphology irregularities. To analyze the changes in
nuclear shape, the perimeter and the area of nuclei is measured in
cells at different passages. This measurement will be carried out
on the midsection of the nucleus by using the overlay/measure
function in the LSM software (Zeiss). To determine the extent of
nuclear lobulation, 50-200 randomly selected nuclei are measured
per passage and the nuclear roundness or contour ratio
(4.times.area/perimeter.sup.2) is calculated. The contour ratio for
a circle is one. As the nucleus becomes more lobulated, this ratio
approaches zero. To calculate the perimeter length and area, the
outline of nuclei is traced with a closed loop drawing tool using
images captured from cells prepared for immunofluorescence with LA
Ab (Anti-lamin A/C Monoclonal Antibody, Unconjugated, Clone JoL2,
MAB3211; Chemicon, Temecula, Calif.). Lamin fluorescence that is
associated with the edge of the lobulations and deep invaginations
that typify the surface of later-passage HGPS cells will be
included. Significance of the resultant observations can be
measured using, for instance, a two-tailed Student t test.
Example 8
Animals Engineered to Express Lamin A/Progerin or Mutants
Thereof
[0267] Mutant or transgenic organisms that under-express or
over-express lamin A protein are useful for research and
characterization, for instance, the characterization of FTIs and
potential inhibitory molecules. They also allow insight into the
physiological and/or pathological role of lamin A in a healthy
and/or pathological organism, for instance in characterization of
aging and aging-related diseases and conditions, including progeria
and atherosclerosis. These mutants are "genetically engineered,"
meaning that information in the form of nucleotides has been
transferred into the mutant's genome at a location, or in a
combination, in which it would not normally exist. Nucleotides
transferred in this way are said to be "non-native." For example, a
non-LMNA promoter inserted upstream of a native LMNA encoding
sequence would be non-native. An extra copy of an LMNA gene on a
plasmid, transformed into a cell, would be non-native.
[0268] Mutants may be, for example, produced from mammals, such as
mice, that either over-express lamin A or under-express lamin A, or
that do not express lamin A at all, or that express a mutant form
of lamin A (such as the splice variant described herein), that
express one or more of these proteins under control of an inducible
promoter, and so forth. Over-expression mutants can be made by
increasing the number of LMNA genes in the organism, or by
introducing an LMNA gene into the organism under the control of a
constitutive or inducible or viral promoter such as the mouse
mammary tumor virus (MMTV) promoter or the whey acidic protein
(WAP) promoter or the metallothionein promoter. Mutants that
under-express lamin A may be made by using an inducible or
repressible promoter, or by deleting the LMNA gene, or by
destroying or limiting the function of the LMNA gene, for instance
by disrupting the gene by transposon insertion.
[0269] A gene is "functionally deleted" when genetic engineering
has been used to negate or reduce gene expression to negligible
levels. When a mutant is referred to in this application as having
the LMNA gene altered or functionally deleted, this refers to the
LMNA gene and to any ortholog of this gene. When a mutant is
referred to as having "more than the normal copy number" of a gene,
this means that it has more than the usual number of genes found in
the wild-type organism, e.g., in the diploid mouse or human.
[0270] A mutant mouse or other animal over-expressing normal or
mutant lamin A may be made by constructing a plasmid having an LMNA
encoding sequence driven by a promoter, such as the mouse mammary
tumor virus (MMTV) promoter or the whey acidic protein (WAP)
promoter. This plasmid may be introduced into mouse oocytes by
microinjection. The oocytes are implanted into pseudopregnant
females, and the litters are assayed for insertion of the
transgene. Multiple strains containing the transgene are then
available for study.
[0271] WAP is quite specific for mammary gland expression during
lactation, and MMTV is expressed in a variety of tissues including
mammary gland, salivary gland and lymphoid tissues. Many other
promoters might be used to achieve various patterns of expression,
e.g., the metallothionein promoter.
[0272] An inducible system may be created in which the subject
expression construct is driven by a promoter regulated by an agent
that can be fed to the mouse, such as tetracycline. Such techniques
are well known in the art.
[0273] In particular, one example transgenic animal is a mouse
model of HGPS, duplicating one of the G608G mutations. The mouse
sequence is perfectly identical at this region of the coding
sequence, so this would produce the same kind of consequence for
lamin A as in the human.
[0274] In addition to knock-out systems, it is also beneficial to
generate "knock-ins" that have lost expression of the wildtype
protein but have gained expression of a different, usually mutant
form of the same protein.
[0275] By way of example, the dominant mutant lamin A protein
(progerin) provided herein, or any of the other mutant lamin A,
progerin, or progerin-like proteins described herein, can be
expressed in a knockout background, such as a mutant mouse that has
been rendered defective or selectively defective (e.g., inducibly
knocked-out) for LMNA expression, in order to provide model systems
for studying the effects of treatment with FTIs or potential
farnesyltransferase inhibitory compounds. In particular
embodiments, the resultant knock-in organisms provide systems for
studying aging, arteriosclerosis, and/or HGPS-like conditions.
[0276] Those of ordinary skill in the relevant art know methods of
producing knock-in organisms. See, for instance, Rane et al. (Mol.
Cell Biol., 22: 644-656, 2002); Sotillo et al. (EMBO J., 20:
6637-6647, 2001); Luo et al. (Oncogene, 20: 320-328, 2001);
Tomasson et al. (Blood, 93: 1707-1714, 1999); Voncken et al.
(Blood, 86: 4603-4611, 1995); Andrae et al. (Mech. Dev., 107:
181-185, 2001); Reinertsen et al. (Gene Expr., 6: 301-314, 1997);
Huang et al. (Mol. Med., 5: 129-137, 1999) by way of example.
Example 9
Mouse Systems for Testing FTIs
[0277] Mice from positive progerin-expressing lines, or lines
expressing other abnormal lamin proteins, can be treated with
differing doses of FTIs and compared with controls for the
prevention or amelioration of any phenotypic changes (for instance,
growth changes, ability to reproduce, hair loss, skin changes,
vascular changes, and nuclear blebbing).
[0278] One specific dosing regimen for examining FTI effects in
transgenic mice is as follows, administered (e.g., orally, such as
by oral gavage) over differing time periods, for instance 10 days
to 3 weeks:
TABLE-US-00002 R115777 SCH66336 10 mg/kg BID 10 mg/kg BID 25 mg/kg
BID 25 mg/kg BID 50 mg/kg BID 50 mg/kg BID 100 mg/kg BID 100 mg/kg
BID
Alternatively, an FTI can be provided in food. By way of example,
an FTI (such as FTI R115777) can be provided in by way of animal
(e.g., mouse) feed at doses as follows: 450 mg/kg of feed, 150
mg/kg of feed, and 0 mg/kg of feed (as a negative control). One of
ordinary skill will recognize that all of these dosages are
examples only, and can be varied.
Example 10
Vascular Disease Process in Hutchinson-Gilford Progeria
Syndrome
[0279] HGPS skin fibroblasts displayed disruption of nuclear
architecture, premature senescence, apoptosis, decreased hyaluronan
secretion and decreased matrix metalloproteinase secretion. The
disruption of nuclear architecture and apoptosis has been examined
in vascular smooth muscle cells (VSMC) transfected with progerin.
In addition, by transfecting the progeria minigene into normal VSMC
and endothelial cells (EC), it was shown that these cell types
produce progerin. It was hypothesized that disruption of nuclear
structure and function culminates in premature cell death and cell
senescence of VSMC and EC. It is believed that these features can
be improved or reversed by blocking post-translational processing
of lamin A using farnesyltransferase inhibition, and that
elimination of progerin will restore normal function to progeria
and other laminopathy cells.
[0280] LMNA is a 12 exon gene that initially produces a pre-lamin A
which must be processed both outside of the nucleus via
isoprenylation of a carboxy terminal CAAX motif (SEQ ID NO: 31),
cleavage of the last three amino acids, and methyl esterification,
and inside of the nucleus via proteolysis of its terminal 18 amino
acids to become mature lamin A (Sinensky et al., J Cell Sci 107(Pt
1):61-7, 1994). The deletion in HGPS probably does not affect the
ability of Progerin to localize to the nucleus or to dimerize (as
normal lamin A does), because the components needed for nuclear
localization and dimerization are not deleted. The ability to
isolate progerin from cells (FIG. 15), yet detect lamins only in
the nucleus via anti-lamin A using immunofluorescence (FIG. 19),
supports this hypothesis.
A. Senescence and Apoptosis
[0281] Many of the features of HGPS, such as atherosclerosis and
bony changes, may be at least partially attributed to premature
cellular senescence or apoptosis in vivo. The data demonstrate that
HGPS fibroblasts have less than half of the growth potential and
show premature apoptosis as compared to normal skin fibroblasts
from age-matched or older donors. These investigations have been
extended to HGPS-transformed vascular cell types and senescence and
apoptosis have been addressed in the principal cell types involved
in abnormalities of vasculature (VSMC and EC).
[0282] Cell Culture:
[0283] VSMC and EC were kindly provided by Richard Karas, MD, PhD,
New England Medical Center, Boston, Mass. HGPS and normal
fibroblasts cells were obtained from the Coriell Cell Repositories
and the Progeria Research Foundation.
[0284] Dermal fibroblasts were maintained in MEM with Earl's Salts
(Gibco, 10370-021) (EMEM), 1.times. Pen./Strep. (100 U/ml P, 100
.mu.g/ml S), 2 mM L-Glutamine and 15% fetal bovine serum, under 5%
CO.sub.2. Cell numbers were obtained at every passage using a
hemocytometer and Trypan Blue cell viability staining. VEC were
grown on gelatin (Sigma #G1393) in Medium 199 in Earl BSS with 2 mM
L-glutamine, 0.05-0.1 mg/mL Endothelial Cell Growth Supplement
(Fisher #CB40006B)*, 0.10 mg/ml Heparin (Sigma H3149), 1.times.
Pen/Strep, and 10% fetal bovine serum. VSMC were grown with either
DMEM+10% Fetal Bovine Serum+2 mM L-glutamine (1.times.) and
1.times. Pen/Strep (as above) or Clonetics.RTM. Media Systems SmGM2
media (CC-3182).
[0285] Cell cultures were fed every 2-3 days, and split when
necessary using 0.05% trypsin.
[0286] Assay for Growth Rate:
[0287] Growth rate was assessed by measuring population doubling
times. Population doublings (PD)=log.sub.2(N/N.sub.0), where
N=final cell number and N.sub.0=starting cell number. Therefore,
PD=(log.sub.10N/N.sub.0)/0.301 and PD time=Total incubation
time/PD.
[0288] Senescence-Associated .beta.-Galactosidase (SA-.beta. Gal)
and Cell Cycling:
[0289] Pre-confluent cultured cells were fixed with 3% formaldehyde
and stained with 1 mg/ml 5-bromo-4-chloro-3-indolyl
.beta.-D-galactoside (X-gal), 40 mM citric acid/sodium phosphate
buffer pH 6.0, 5 mM potassium ferrocyanide, 5 mM ferricyanide, 150
mM sodium chloride and 2 mM magnesium chloride. At pH 6.0 the
solution specifically stains senescent cells (cells in G1-like
arrest) blue in the perinuclear region (Dimri et al., Proc Natl
Acad Sci USA. 92:9363-9367, 1995). Exposure to 10
.mu.Ci/ml-.sup.3H-thymidine followed by fixation and development
for silver grains was used to assay cell cycling.
Results and Discussion
[0290] In vitro, fibroblasts normally progress to senescence, and
this is considered a hallmark of "aging" in vitro. Fibroblasts from
older donors senesce more quickly than those from younger donors
(Martin et al., Laboratory Investigation 23:86-92, 1970). There are
conflicting reports on the growth potential of HGPS fibroblasts
(Goldstein, J Inv. Derm. 73:19-23, 1979). Seven different HGPS skin
fibroblast lines have been cultured, and they have senesced
completely, or have reached Phase 3 growth, as indicated by an
increase in doubling time and changes in morphology (Hayflick, J Am
Geriatr Soc 22(1):1-12, 1974), between passages 6 and 18 (Table 1).
This is less than half of the growth potential of normal skin
fibroblasts from age-matched or older donors (Schneider &
Mitsui, Proc. Nat. Acad. Sci. 73:3584-3588, 1976) (FIG. 17). In
general, the senescing HGPS cells are increased in size and show
characteristic intracellular changes associated with senescence,
whereas normal fibroblasts after similar passage number do not.
Another hallmark of senescence is cytoplasmic staining with
SA-.beta. gal (Dimri et al., Proc Natl Acad Sci USA. 92:9363-9367,
1995). A higher percentage of HGPS fibroblasts stained positively
for SA-.beta.gal activity than normal fibroblasts of similar
passage, while the same normal fibroblasts incorporated a much
higher percentage of .sup.3H-thymidine than the HGPS fibroblasts,
indicating increased cell cycling.
TABLE-US-00003 TABLE 1* Percentages of cycling and senescent cells
Normal Control HGPS % PD % PD Passage SA-.beta. % time Passage
SA-.beta. % time # gal Thymidine (h) # gal Thymidine (h) 12 6 80
44.8 9 11 67 132 10 20 90 58.9 13 56 22 236 16 0 85 54.5 16 48 15
431 *each line represents a separate experiment; 200 cells randomly
assayed
B: Nuclear Morphology and Disease Phenotype
[0291] Immunofluorescent Staining for Lamin a and Nuclear
Morphology:
[0292] Cells were seeded into glass coverslips, methanol fixed,
incubated in primary (mouse anti-human lamin A/C monoclonal used at
1:10, MAB3211, Chemicon) and fluorescent secondary (Alexa Fluor 488
goat anti-mouse IgG used at 1:3000, A-11001, Molecular Probes)
antibodies and counter-stained with DAPI. Confocal Microscopy can
also be used to better identify nuclear blebbing.
[0293] Assays for Apoptosis:
[0294] The occurrence of apoptosis in normal versus
HGPS-transformed cultures was determined using an Annexin V binding
assay (Vybrant.RTM. Apoptosis Assay Kit #2, Molecular Probes)
adapted for adherent cell cultures. Alternatively, caspase analysis
may be used for apoptosis analysis, using known protocols.
Results and Discussion
[0295] Alteration of lamin A produces blebbing of nuclei in
cultured HGPS fibroblasts (Eriksson et al., Nature
423(6937):293-298, 2003) and the data show that this nuclear
blebbing increases significantly with passage number. Early
passage, vigorously growing, normal fibroblasts with low levels of
senescence associated .beta.-galactosidase (SA-.beta. gal) staining
(6%; FIG. 17, Point A) had normal, round nuclei (FIG. 19A), a
blebbing rate of 12% (Table 2), and virtually no apoptotic cells
(FIG. 18). This rate increased to 22% blebbing at passage 12. Early
passage, vigorously growing, HGPS fibroblasts with correspondingly
low levels of SA-.beta. gal staining (6%; FIG. 17, Point C)
demonstrated abnormally blebbed nuclei in 11% of cells and this
percentage grew to 28% at passage 12 and then 50% at passage 14,
when growth curves demonstrated almost complete senescence in the
HGPS fibroblasts (Table 2).
TABLE-US-00004 TABLE 2 Percentage of nuclei exhibiting abnormal
morphology (blebbing). passage # cells Condition number counted #
blebs % blebs Normal p9 113 14 12.4% Normal p12 102 22 21.5% HGPS
htert p28 106 17 16.0% HGPS htert p34 111 26 21.6% HGPS p6 120 13
10.8% HGPS p12 125 35 28.0% HGPS p14 150 75 50.0%
[0296] The percentage of cells with nuclear blebbing increased with
passage number in HGPS fibroblasts (Table 2). The potential causal
association of nuclear blebbing and thus nuclear membrane
instability with senescence and apoptosis in HGPS-transformed
vascular cells is being studied further. To understand whether
blebbing precedes or coincides with these events, cultures are
co-stained for senescence or apoptosis and for nuclear morphology
at varying cell passages.
C: ECM and Vascular Disease in HGPS
[0297] There is considerable indication, both in vivo and in vitro,
that HGPS is a disease heavily involving abnormalities in the
extracellular matrix, with increased collagen and elastin
secretion, disorganized dermal collagen, and decreased decorin over
normal controls (reviewed in Davidson et al., Ciba-Found-Symp.
192:81-99, 1995). Extracellular matrix molecules have both
structural and cell signaling functions in skin, bone, and the
cardiovascular system all severely affected in HGPS.
[0298] Reduction in hyaluronic acid (HA)-dependent pericellular
matrices (PCM) (HA-PCM) may be involved in prevention of cellular
senescence by acting as a protective sink for reactive oxygen
species. Reactive oxygen species are produced as a normal
consequence of the aerobic cellular metabolism. However, oxidative
stress induces senescence and apoptosis and is implicated in
etiology of a long list of diseases, including HGPS (Yan et al.,
Biochemical and Biophysical Research Commun. 257:163-167, 1999) and
atherosclerosis (reviewed in Napoli et al., J. Cell. Biochem.
82:674-682, 2001), as well as the process of normal aging (Floyd et
al., Exp. Gerontol. 36:619-640, 2001). Abnormalities in the
extracellular matrix (ECM) may be key components to disease process
in HGPS. Hyaluronan-cell interactions promote cell growth and
survival signaling pathways. Published microarray analyses (Csoka
et al., Aging Cell 3:235-43, 2004) of HGPS fibroblasts and our
protein data show dramatic decreases in stromelysin (MMP-3) over
normal control fibroblasts.
[0299] MMP's are key components of ECM restructuring for
angiogenesis. By measuring the size of HA-PCM and MMP-3 in VSMC and
EC, a correlation between premature cellular senescence and
decreased matrix assembly in HGPS cultures can be
substantiated.
[0300] HA-PCM Assay:
[0301] HA-PCM size was analyzed using a well-established particle
exclusion assay (Knudson & Toole, Dev Biol 112(2):308-318,
1985). Briefly, a suspension of formalin-fixed erythrocytes (0.8 ml
of 10.sup.8 cells/ml in 0.1% BSA in PBS) was added to a 35 mm dish
of sub-confluent fibroblasts, settled, and photographed using a 4.3
megapixel digital camera (VersaCam Digital Imaging Outfit, Chestnut
Hill, Mass.). Morphometric analysis of the HA-PCMs was determined
using Scion Image Beta 4.0.2 software (Scion Corp., Frederick, Md.)
for analysis of cell perimeter and matrix perimeter.
[0302] MMP-3 Analysis:
[0303] MMP-3 can be assayed from confluent culture conditioned
media using a standard ELISA kit (Amersham MMP3 ELISA kit
(RPN2613)).
Results and Discussion
[0304] Hyaluronan production by normal and HGPS fibroblasts was
measured. Hyaluronan content in pre-senescent HGPS and age-matched
normal control fibroblasts was analyzed using
fluorophore-associated carbohydrate electrophoresis analysis,
revealing a 70% decrease in both the cell layer and culture medium
over age matched control cultures.
TABLE-US-00005 TABLE 3 Hyaluronan Content in Fibroblast Cultures
(ng/ml)* Cell Layer Media Layer Normal Pre-senescent 1005 572 HGPS
Senescent 136 343 *age and passage - matched (n = 2); ELISA-like
assay
[0305] Dermal fibroblasts, VSMC and EC exhibit highly hydrated
HA-PCMs that can be visualized indirectly by their ability to
exclude particles (see methods section). Preliminary data suggested
that the HA-PCM thickness of senescent HGPS fibroblasts was 12-fold
decreased over age matched pre-senescent HGPS fibroblasts and
22-fold decreased over normal dermal fibroblasts (Table 4).
TABLE-US-00006 TABLE 4 Mean HA-PCM radius in cultured fibroblasts
(.mu.m)* Normal Pre-senescent 6.7 Normal Senescent 1.5 Progeria
Pre-senescent 3.7 Progeria Senescent 0.3 *1 .times. 10.sup.4 cells
were plated per 35 mm dish, and HA-PCM radii were measured on 25
cells randomly chosen prior to addition of fixed red blood
cells.
[0306] The data substantiate a correlation between premature
cellular senescence and decreased matrix assembly in HGPS
fibroblast cultures. Large polymeric hyaluronan protects against
oxidative stress by absorbing free radicals and inhibiting
pro-inflammatory cytokine formation (reviewed in Neumann et al.,
FEBS Letters 453:283-7, 1999). This evidence suggests that changes
in hyaluronan (previously named hyaluronic acid or hyaluronate)
precede onset of standard senescent characteristics in HGPS
fibroblasts but resemble changes that also occur during senescence
of normal fibroblasts. These results are of broad interest because
hyaluronan is involved in the development of atherosclerotic
lesions and has also been found to play a role in cell survival
signaling cascades.
[0307] Matrix Metalloproteinase-3 (MMP-3) has been associated with
cellular senescence (Parrinello et al., J. Cell Sci. 118:485-496,
2005), atherosclerotic plaque formation (Doherty et al., Mayo Clin.
Sci. 79:197-210, 2004), and HGPS (Csoka et al., Aging Cell
3:235-243, 2004; Ly et al., Science 287:2486-2492, 2000). A
complete absence of MMP-3 was observed in HGPS dermal fibroblast
cultures, as compared to normal control dermal fibroblast cultures
(FIG. 20). This molecule therefore serves as a biomarker of
cellular disease in HGPS, which would be normalized with effective
treatment of disease process, and helps to elucidate the mechanism
of disease in HGPS.
D: Vascular Changes and HGPS
[0308] Atherosclerosis in HGPS is a diffuse process, involving the
large vessels, the coronary arteries, and distal vasculature. To
date there have been fewer than 20 autopsies described on children
with HGPS (reviewed in Stehbens et al., Cardiovasc Pathol
10(3):133-136, 2001). These studies note focal atherosclerotic
plaques throughout large and small arteries, including all coronary
artery branches, interstitial fibrosis and stenosis. Plaques are
markedly calcified with cholesterol crystals evident. Microscopy
reveals thickened and almost acellular hyaline fibrosis. Notably,
two autopsies report a paucity of medial smooth muscle cells, with
acellular fibrous tissue predominating in the media. Intimal and
medial collagen have been noted as disorganized and of small
diameter. However, a major problem with studying autopsy specimens
is that we are observing the end stage of a complicated process
(Zhang et al., Am J Pathol. 143:496-506, 1993).
[0309] Calcification of both vascular intima and media are early
events in the development of atherosclerotic plaques in aging
individuals and those with diabetes (Cooper et al., Am J Hypertens.
14:475-86, 2001). Apoptosis occurs in vascular smooth muscle cells
(VSMC) prior to the development of calcification (Proudfoot et al.,
Circulation 106:3044-50, 2002), and may even be required for
calcification to occur. Vascular calcification is implicated in
both pathological and in vitro studies as a requisite event in
plaque formation. Therefore apoptosis may be a key element to
development of disease in HGPS. Premature apoptosis occurs in HGPS
fibroblasts (FIG. 18).
E: Creating HGPS in Vascular Cells:
[0310] One way to begin understanding the initial phases of
atherosclerosis in HGPS is to study the VSMC and EC at initial
phases of disease. This can be achieved by introducing the HGPS
gene, or constructs expressing similar mutant forms of lamin A,
into normal VSMC and EC. If progeric VSMCs have limited capacity
for proliferation, as do fibroblasts (FIG. 15), then the clonal and
proliferative requirements of VSMC in plaque initiation may lead to
early senescence or apoptosis of VSMC and eventually result in the
lack of VSMC seen at autopsy.
[0311] Constructs were created that allowed VSMC and EC to
demonstrate natural splicing capabilities, thus demonstrating that
these cell types do express progerin in vivo.
[0312] Recombinant DNA Constructs Using Site Directed
Mutagenesis:
[0313] Briefly, RT-PCR products of LMNA were generated using RNA
from normal dermal fibroblasts and the GC-rich protocol for
SUPERSCRIPT.RTM. reverse transcriptase First-Strand Synthesis
System for RT-PCR (Invitrogen) with Platinum Taq High Fidelity and
PCRx Enhancer System (Invitrogen). The LMNA cDNA was then cloned
using a pCR2.1-TOPO cloning vector (Invitrogen) and One Shot TOP10
Chemically Competent E. coli (Invitrogen).
[0314] Normal Lamin A:
[0315] The normal lamin A cDNA was cloned via RT-PCR from RNA
extracted from normal human dermal fibroblast line GM00969D
(Coriell Repositories). The cDNA was inserted into the pEGFP-C
mammalian expression vector (Clontech) at the EcoRI site (FIG.
14).
[0316] Progerin:
[0317] The mutant HGPS splice variant of lamin A was created from
the normal lamin A construct via site directed mutagenesis
(Stratagene). Bases 1818 through 1968 were deleted from exon 11
producing a cDNA that codes for the mutant protein "progerin" with
a GFP fused to its N terminus (FIG. 14).
[0318] Minigene:
[0319] A third construct was created, which contained the intron
between exon 11 and 12 as well as the c-to-t point mutation in
codon 608 in exon 11 (G608G) responsible for HGPS. The presence of
this intron allows a cell expressing the construct to recognize the
cryptic splice site caused by the point mutation, and splice both
the normal and abnormal lamin A products (FIG. 14).
[0320] Quantitative PCR:
[0321] Total RNA was extracted from cells harvested via
trypsinization using the RNEASY.RTM. Mini RNA isolation Kit
(Qiagen). Five hundred nanograms (500 ng) of each mRNA template was
reverse-transcribed with an oligo(dT) primer using the SUPERSCRIPT
II.RTM. reverse transcriptase first strand cDNA synthesis kit
(Invitrogen). Quantitative PCR was then carried out on a Stratagene
MX 4000 apparatus using the QUANTITECT.RTM. SYBR Green PCR analysis
kit from Qiagen with the following primers: Forward (SEQ ID NO: 9)
and Reverse (SEQ ID NO: 10).
[0322] Vascular cells (EC, FIG. 16A; coronary artery SMC, FIG. 16B;
radial artery SMC, FIG. 16C) were electroporated and exposed to
either a progerin minigene or LMNA-lamin A minigene. These cell
types were demonstrated to splice the minigene; the amplified,
processed transcript (489 base pairs) is indicated with an
arrow.
[0323] Protein Electrophoresis and Western Blotting:
[0324] Protein isolated from cultures are probed using primary
mouse anti-human lamin A/C monoclonal used at 1:200, MAB3211,
Chemicon, followed by secondary antibody (sheep anti-mouse Fab
IgG-HRP conjugate, 1:3000, Amersham) and visualized for band
intensity using the Eagle Eye II Still Video System
(Stratagene).
Example 11
Inhibiting Farnesylation of Progerin Prevents the Characteristic
Nuclear Blebbing of Hutchinson-Gilford Progeria Syndrome
[0325] It was hypothesized that retention of the farnesyl group
causes progerin to become permanently anchored in the nuclear
membrane, disrupting proper nuclear scaffolding and causing the
characteristic nuclear blebbing seen in HGPS cells. Progerin's
central rod domain then allows dimerization with mature,
nonfarnesylated lamin A and assembly into a multiprotein complex,
resulting in dominant negative disruption of the nuclear
scaffolding and underlying heterochromatin, and leading to the
characteristic nuclear blebbing seen in HGPS (Goldman et al., Proc.
Natl. Acad. Sci. USA 101:8963-8968, 2004). It was further
hypothesized that farnesyltransferase inhibitors (FTIs) would
inhibit the formation of progerin, and that decreasing the amount
of this aberrant protein could potentially improve disease status
in HGPS and other laminopathies.
[0326] In this study, the ability of both genetic mutation and
pharmacological treatment to prevent the dysmorphic nuclear
phenotype seen in HGPS was examined. The terminal CSIM sequence
(SEQ ID NO: 32) in progerin was mutated to SSIM (SEQ ID NO: 33), a
sequence that cannot be farnesylated.
[0327] Material included in this example was also published as
Capell et al., Proc. Nat. Acad. Sci. 102:12879-12884, Sep. 6, 2005,
which is incorporated herein by reference in its entirety.
Materials and Methods
[0328] Constructs and Mutagenesis.
[0329] The pEGFP-myc-LA vector (referred to here as wild-type lamin
A-CSIM) and the L.DELTA.450 (referred to here as progerin-CSIM)
vector were created as previously described (Goldman et al., Proc.
Natl. Acad. Sci. USA 101:8963-8968, 2004) and encode green
fluorescent protein (GFP)-tagged lamin A fusion proteins. The
wild-type lamin A-CSIL, progerin-CSIL, wild-type lamin A-SSIM, and
progerin-SSIM mutations were created by site-directed mutagenesis
using the following oligos: for CSIL (SEQ ID NO: 13) and for SSIM
(SEQ ID NO: 11) (QUIKCHANGE.RTM. II XL Site-Directed Mutagenesis
Kit, Stratagene).
[0330] Cell Culture.
[0331] Cell lines used were the normal human fibroblast line,
AG06299, and HGPS fibroblast lines: AG06917, AG11513, and AG11498
(obtained from the NIA Aging Cell Culture Repository, Coriell
Institute for Medical Research, Camden, N.J.). Fibroblasts were
cultured in minimal essential medium (MEM; Invitrogen/Gibco)
supplemented with 15% FBS (HiClone), 2 mM L-glutamine, penicillin
(50 U/ml) and streptomycin (50 mg/ml). HeLa and HEK-293 cell lines
were cultured in DMEM (Invitrogen/Gibco) supplemented with 10%
fetal bovine serum and antibiotics.
[0332] Transient Transfections.
[0333] Approximately 25,000 cells were plated per chamber of
4-chamber slides (Lab-Tek, Nalge Nunc International, #154526).
After twenty-four hours, HeLa and HEK-293 cell lines were
transiently transfected with 0.8 .mu.g of each construct using
LIPOFECTAMINE.RTM.2000 transfection reagent (Invitrogen) under
standard conditions.
[0334] FTI Treatment.
[0335] At the time of transient transfection, HeLa cells were
treated with one dose of 0, 0.5, 1.0, or 2.0 .mu.M of the selective
FTI lonafarnib [Sarasar, SCH66336, (Schering-Plough)]. HEK-293
cells were treated with one dose of 0, 1, 2, 5, or 10 .mu.M of the
selective FTI L-744832 (Biomol). In addition, NIH-3T3 cells were
treated with 5 .mu.M of FTI-2153, which is highly selective for
FTase, or GGTI-2166, which is highly selective for GGTase I (kind
gifts of Said M. Sebti and Andrew D. Hamilton). These FTIs have the
same mechanism of action and can be used interchangeably to inhibit
protein farnesylation in vitro. However, only lonafarnib is a
clinical candidate. Normal and HGPS fibroblasts were treated with
one daily dose of 0, 0.5, 1.0, or 2.0 .mu.M of lonafarnib for 3
days.
[0336] GFP Localization and Fluorescence Microscopy.
[0337] Forty-eight hours following transient transfection, the HeLa
and HEK-293 cells were visualized for GFP localization using an
LSM510 confocal microscope (Zeiss). Following three-day treatment
with lonafarnib, HGPS fibroblasts were washed two times with
phosphate-buffered saline (PBS) (pH 7.2) and fixed for 10 minutes
at room temperature with 1% paraformaldehyde in PBS. Following
three washes with PBS, the cells were permeabilized for 5 minutes
at room temperature with 0.5% Triton X-100 in PBS and blocked with
5% horse serum for 30 minutes at room temperature. Cells were then
incubated for one hour at room temperature with the primary
antibody diluted in blocking solution, a polyclonal mouse
anti-human lamin A/C at 1:10 (mAb3211, Chemicon). Three more washes
with PBS were then followed by incubation with the secondary
antibody, Alexa 488-conjugated donkey anti-mouse IgG (Molecular
Probes), for 45 minutes and three additional PBS washes. Slides
were then mounted with mounting medium containing DAPI (Vector
Laboratories).
[0338] Mobility Shift Experiments.
[0339] NIH-3T3 cells, grown in DMEM-H (Gibco/Invitrogen)
supplemented with 10% GCS at 37.degree. C. in 10% CO.sub.2, were
transiently transfected with either empty vector, progerin-CSIM or
progerin-SSIM using LIPOFECTAMINE.RTM.-Plus transfection reagent
(Invitrogen) according to the manufacturer's instructions.
Transfected cells were treated for 48 hours with vehicle (DMSO) or
inhibitors of prenylation (5 .mu.M FTI-2153, 5 .mu.M GGTI-2166 or
both). Cells were lysed directly in 2.times. Laemmli loading buffer
and total cell lysates were resolved on 8% SDS PAGE. Proteins were
transferred to Immobilon PVDF (Millipore), blotted with anti-GFP
mAb (clone B34, Covance) and visualized using SUPERSIGNAL.TM.
enhanced chemiluminescence (Pierce).
[0340] Morphometric Analysis.
[0341] To examine the overall percentage of blebbed cells in cells
with different amounts and lengths of FTI treatment, 200 nuclei
were classified as either blebbed, if they contained two or more
lobulations, or not blebbed, as previously described (Goldman et
al., Proc. Natl. Acad. Sci. USA 101:8963-8968, 2004). These
classifications were done by three independent observers, all
blinded to the specifics of the cells being examined, followed by
averaging three independent data sets. Armitage's trend test
(Armitage, Biometrics 11:375-386, 1955) was used to test for a
dose-dependent response for each cell type.
Results
[0342] While both B-type lamins and lamin A are farnesylated and
carboxymethylated, unique to lamin A is a second cleavage step that
occurs inside the cell nucleus causing the removal of an additional
15 C-terminal amino acids from the mature protein, including the
farnesylated cysteine. This final cleavage step, and the resulting
loss of the farnesyl anchor, is believed to release prelamin A from
the nuclear membrane and allow it to be inserted into the nuclear
lamina. In HGPS, although preprogerin can be farnesylated, its
internal deletion of amino acids 606-656 removes the endoprotease
recognition site necessary for executing the final cleavage step
(FIG. 21).
[0343] We sought to examine the ability of CAAX box (SEQ ID NO: 31)
mutants or FTIs to block the nuclear blebbing that is the cellular
hallmark of the HGPS phenotype. To examine the effects of
inhibiting all prenylation, missense mutations of the cysteine
residue of the CSIM motifs (SEQ ID NO: 32) of wild-type lamin A and
progerin were generated (designated SSIM). These constructs, marked
for visualization by an amino-terminal GFP fusion, were transfected
into HeLa cells and imaged after 48 hours. Without prenylation, all
of the prelamin A was relocated from its normal location at the
nuclear periphery into nucleoplasmic aggregates, and there was no
progerin-induced nuclear blebbing (FIG. 22). Observations by
confocal microscopy suggested that these nucleoplasmic aggregates
were distributed throughout the nucleoplasm. Mutating the CSIM
terminal sequence (SEQ ID NO: 32) to CSIL was predicted to favor
geranylgeranylation by GGTase I rather than farnesylation by FTase
(Cox & Der, Curr. Opin. Pharmacol. 2:388-393, 2002) and
therefore generate an FTI-resistant form of the protein. In this
case, progerin-induced nuclear blebbing persisted (FIG. 22)
indicating that the persistent modification of any prenoid group
promotes the progerin phenotype. Similar results for all of these
constructs were obtained with HEK-293 cells and NIH-3T3 cells.
[0344] We then explored the effect of treating HeLa cells
transiently transfected with wild-type lamin A-CSIM, progerin-CSIM,
progerin-SSIM, and progerin-CSIL with the clinical candidate FTI
lonafarnib (Ganguly et al., Curr. Med. Chem. 8:1419-1436, 2001).
Following a single dose of 0, 0.5, 1.0, 2.0 .mu.M at the time of
transfection, cells were visualized 48 hours later (FIGS. 23 and
24). In the wild-type lamin A-CSIM transfected cells, cell counting
demonstrated that the percentage of nuclear blebbing was unchanged
despite treatment, remaining at approximately 5%, which is the
typical blebbed percentage for normal wild-type cells (Goldman et
al., Proc. Natl. Acad. Sci. USA 101:8963-8968, 2004). In contrast,
those cells transfected with the progerin-CSIM constructs showed a
dramatic dose-dependent response (p<0.0001), with the nuclei
approaching normal blebbing percentages with a single dose of 2.0
.mu.M of lonafarnib. The progerin-CSIL mutant, predicted to be
geranylgeranylated, was completely resistant to treatment with the
FTI, evidence that it is inhibition of the farnesylation of
progerin, and not of any other endogenous farnesylated protein,
that is responsible for the improvement in nuclear phenotype seen
with FTI treatment. As expected, there was no difference seen with
FTI treatment of the progerin-SSIM mutant, as prenylation had
already been completely inhibited. Similar results for all of these
constructs were obtained with HEK-293 and NIH-3T3 cells using the
FTIs L-744832 or FTI-2153 in doses ranging from 1.0 .mu.M to 10
.mu.M.
[0345] Next, the effect of FTIs on nuclear blebbing was examined in
cells from individuals with HGPS, in this case dermal fibroblasts.
Cultured fibroblasts from three HGPS patients and one unaffected
mother were treated for three days with a once daily dose of
lonafarnib. In order to observe nuclear morphology at the end of
treatment, nuclei were stained with an antibody for lamin A and C
(FIGS. 25 and 26). On day three, all three HGPS fibroblast lines
demonstrated a significant dose-dependent reduction in nuclear
blebbing that was unaffected by either patient age or cell passage
number (p<0.0001).
[0346] If treatment with FTIs is to be considered a viable option
for HGPS, it is important to determine whether authentic progerin,
like K-Ras and some other farnesylated proteins (Zhang et al., J.
Biol. Chem. 272:10232-10239, 1997) could still be
geranylgeranylated in the presence of FTIs and therefore resistant
to FTI treatment. To explore this possibility, a series of
experiments were performed examining the migration of progerin on a
highly resolving gel when treated with either an FTI, a
geranylgeranyltransferase inhibitor (GGTI), or a combination of
both drugs. The appearance of a shift in mobility to a slower
migrating form is a standard method of demonstrating the ability of
FTIs to prevent the processing of farnesylated proteins. Treatment
with GGTI alone had no effect on the mobility of progerin (FIG.
27), demonstrating that progerin is not normally
geranylgeranylated. However, GGTI did completely shift the mobility
of progerin-CSIL whereas FTI had no effect, confirming that this
mutant was processed as expected. Importantly, progerin treated
with either an FTI or with FTI+GGTI migrated identically with
vehicle-treated, completely unprocessed progerin-SSIM mutant,
demonstrating that the processing of progerin can be completely
inhibited by FTI treatment alone, and that progerin is not
alternatively geranylgeranylated when farnesylation is
inhibited.
Discussion
[0347] Farnesylation, a post-translational modification involving
the addition of a 15-carbon isoprene moiety, was first implicated
as a potential anti-cancer target when it was discovered that the
oncoprotein, Ras, which has been estimated to be involved in up to
30% of all human cancers (Bos, Cancer Res. 50:1352, 1989), required
farnesylation for its function (Hancock et al., Cell 57:1167, 1989;
Casey et al., Proc Natl Acad Sci USA., 86:8323, 1989; Schafer et
al., Science 245:379, 1989). With the subsequent purification of
the FTase enzyme (Reiss et al., Cell 62:81-88, 1990), a vigorous
research effort in the pharmaceutical industry has identified and
developed a number of small molecule compounds that potently and
selectively inhibit farnesyltransferase (Sebti & Der, Nat. Rev.
Cancer 3:945-951, 2003). Two of these drugs (lonafarnib/SCH66336
from Schering Plough and tipifarnib/R115777 from Johnson and
Johnson) have entered Phase III trials and have been well
tolerated, including in trials involving children (Doll et al.,
Curr. Opin. Drug Discovery Dev. 7:478-486, 2004). Lonafarnib
competes with protein substrates for binding to the
farnesyltransferase enzyme (Bishop et al., J. Biol. Chem.
270:30611-30618, 1995).
[0348] Similar to Ras, the lamin A precursor is also farnesylated,
with farnesylation serving as a required step to both insert
prelamin A into the nuclear membrane as well as to allow for the
two downstream cleavage steps which complete the processing of
lamin A (Beck et al., J. Cell Biol. 110:1489-1499, 1990). With the
knowledge that the single C to T base change seen in nearly all
cases of HGPS created a cryptic splice site and thus deleted the
normal second endoproteolytic cleavage site in the lamin A
processing pathway, it was hypothesized that progerin was forced to
retain its farnesyl group and could not therefore dissociate itself
from the nuclear membrane. With other members of the nuclear lamina
also potentially becoming trapped in complexes with the
mislocalized progerin, a mechanistic connection between this
permanently farnesylated state and the striking nuclear blebbing
and disrupted nuclear architecture seen in HGPS cells was proposed,
and the possibility of preventing or reversing this phenotype
through farnesyltransferase inhibitors was raised (Eriksson et al.,
Nature 423:293-298, 2003).
[0349] It was hypothesized that FTIs would reduce the dominant
negative effect of mature progerin on nuclear membrane structure.
This pharmacologic effect would not, however, be specific for
progerin; FTIs would also decrease levels of wild-type lamin A,
increase the levels of unfarnesylated prelamin A, and no doubt
affect the posttranslational processing of dozens of other
farnesylated proteins. Although total cellular lamin A would be
expected to decrease, it was hypothesized that the net effect could
still be beneficial, as the abnormal splice that leads to progerin
is produced rather inefficiently by the mutant allele, and so
progerin is present in much lower quantities than wild-type lamin
A. Furthermore, only a small amount of mature lamin A is necessary
for proper nuclear envelope assembly (Lourim & Krohne, J. Cell
Biol. 123:501-512, 1993). In support of this idea, clinical trials
using FTIs demonstrate little toxicity, even when levels of
unfarnesylated prelamin A are significantly raised (Taveras et al.,
Curr. Top. Med. Chem. 3:1103-1114, 2003). Complete absence of lamin
A, however, leads to serious consequences and disease (Sullivan et
al., J. Cell Biol. 147:913-920, 1999).
[0350] The possibility that progerin would be geranylgeranylated in
the presence of FTIs was also explored. This outcome, if found,
might limit the utility of this approach. In the presence of FTIs,
both oncogenic K-Ras and N-Ras serve as alternative substrates for
the related enzyme geranylgeranyltransferase I (GGTase I), thus
remaining biologically active and fully capable of signal
transduction and malignant transformation (Ganguly et al., Curr.
Med. Chem. 8:1419-1436, 2001; Whyte et al., J. Biol. Chem.
272:14459-14464, 1997; Rowell et al., J. Biol. Chem.
272:14093-14097, 1997; James et al., Proc. Natl. Acad. Sci. USA
93:4454-4458, 1996). Thus, although H-Ras does not undergo
alternative prenylation when FTase activity is blocked, FTIs are
not effective inhibitors of the two Ras isoforms most commonly
mutationally activated in human tumors, compromising the
effectiveness of FTIs in the treatment of cancer.
[0351] Fortunately, data reported here confirm that such
alternative isoprenylation does occur to any detectable degree with
progerin, the processing of which is fully inhibited by FTI alone.
These results indicate that farnesylated progerin is responsible
for the dysmorphic nuclear morphology seen in HGPS, as inhibition
of farnesylation via both genetic mutation and pharmacological
intervention prevents nuclear blebbing and redistributes the mutant
progerin protein from the nuclear membrane into nucleoplasmic
aggregates.
[0352] Beyond the prevention of this nuclear phenotype in
transfected cells caused by ectopically expressed progerin, the
ability of lonafarnib to cause a significant reduction in the
percentage of blebbed nuclei seen in HGPS fibroblasts, even at high
passage numbers, is encouraging evidence of its therapeutic
potential against endogenous progerin. Perhaps the removal of
farnesylated progerin, whether through gene expression changes or
through the actual mechanical stabilization of the nuclear
scaffolding, would cause the cells that are most prone to damage in
HGPS to be rescued. In support of this idea is recent work showing
that by simply reducing levels of farnesylated prelamin A by 50%,
the nuclear blebbing and progeria-like phenotype seen in
ZMPSTE24-deficient mice could be eliminated (Fong et al., Proc.
Natl. Acad. Sci. USA 101:18111-18116, 2004).
[0353] The results provided herein support the hypothesis that it
is the permanently farnesylated state of progerin that allows it to
exert its dominant negative effects and cause the devastating
effect on nuclear structure and function. Further, it has been
demonstrated herein that FTIs are capable of reversing this nuclear
phenotype. Since FTIs are currently under evaluation in Phase III
clinical trials as anti-cancer drugs (Sebti & Der, Nat. Rev.
Cancer 3:945-951, 2003), there is considerable patient information
on FTI activity and toxicity. Therefore, FTIs may be a feasible
therapeutic approach for HGPS.
[0354] This disclosure provides evidence that a farnesyltransferase
inhibitor can reverse or inhibit cellular effects caused by the
expression of progerin, or caused by influence of lamin A on
atherosclerosis and aging. The disclosure further provides methods
and compositions that exploit this discovery in order to treat or
ameliorate HGPS and other laminopathies, as well as cellular aging
and atherosclerosis. It will be apparent that the precise details
of the methods and compositions described may be varied or modified
without departing from the spirit of the described invention. We
claim all such modifications and variations that fall within the
scope and spirit of description and the claims below.
Sequence CWU 1
1
34122DNAArtificial sequenceSynthetic Oligonucleotide 1agcagtctct
gtccttcgac cc 22222DNAArtificial sequenceSynthetic Oligonucleotide
2cttccacctc ccacctcatt cc 22327DNAArtificial sequenceSynthetic
Oligonucleotide 3gtggaggagg tggatgagga gggcaag 27430DNAArtificial
sequenceSynthetic Oligonucleotide 4tggagttgcc caggcggtag gagcgggtga
30530DNAArtificial sequenceSynthetic Oligonucleotide 5tcacccgctc
ctaccgcctg ggcaactcca 30626DNAArtificial sequenceSynthetic
Oligonucleotide 6ctgagggctg gaccgcatct ggggac 26722DNAArtificial
sequenceSynthetic Oligonucleotide 7catgggcaat tggcagatca ag
22823DNAArtificial sequenceSynthetic Oligonucleotide 8catgccttgc
aaaatggcgt tac 23918DNAArtificial sequenceSynthetic Oligonucleotide
9tcaggagccc agcgaccc 181017DNAArtificial sequenceSynthetic
Oligonucleotide 10ttctttggca agccccc 171138DNAArtificial
sequenceSynthetic Oligonucleotide 11ccagagcccc cagaactcaa
gcatcatgta atctagag 381238DNAArtificial sequenceSynthetic
Oligonucleotide 12ctctagatta catgatgctt gagttctggg ggctctgg
381340DNAArtificial sequenceSynthetic Oligonucleotide 13ccccagaact
gcagcatctt ataatctaga gtcgacggta 401440DNAArtificial
sequenceSynthetic Oligonucleotide 14taccgtcgac tctagattat
aagatgctgc agttctgggg 401538DNAArtificial sequenceSynthetic
Oligonucleotide 15cttagagccc ccagaactca agcatcatgt aatctggg
381638DNAArtificial sequenceSynthetic Oligonucleotide 16cccagattac
atgatgcttg agttctgggg gctctaag 381738DNAArtificial
sequenceSynthetic Oligonucleotide 17cccagaactg cagcatctta
taatctggga cgggatcc 381838DNAArtificial sequenceSynthetic
Oligonucleotide 18ggatcccgtc ccagattata agatgctgca gttctggg
38191995DNAHomo sapiens 19atggagaccc cgtcccagcg gcgcgccacc
cgcagcgggg cgcaggccag ctccactccg 60ctgtcgccca cccgcatcac ccggctgcag
gagaaggagg acctgcagga gctcaatgat 120cgcttggcgg tctacatcga
ccgtgtgcgc tcgctggaaa cggagaacgc agggctgcgc 180cttcgcatca
ccgagtctga agaggtggtc agccgcgagg tgtccggcat caaggccgcc
240tacgaggccg agctcgggga tgcccgcaag acccttgact cagtagccaa
ggagcgcgcc 300cgcctgcagc tggagctgag caaagtgcgt gaggagttta
aggagctgaa agcgcgcaat 360accaagaagg agggtgacct gatagctgct
caggctcggc tgaaggacct ggaggctctg 420ctgaactcca aggaggccgc
actgagcact gctctcagtg agaagcgcac gctggagggc 480gagctgcatg
atctgcgggg ccaggtggcc aagcttgagg cagccctagg tgaggccaag
540aagcaacttc aggatgagat gctgcggcgg gtggatgctg agaacaggct
gcagaccatg 600aaggaggaac tggacttcca gaagaacatc tacagtgagg
agctgcgtga gaccaagcgc 660cgtcatgaga cccgactggt ggagattgac
aatgggaagc agcgtgagtt tgagagccgg 720ctggcggatg cgctgcagga
actgcgggcc cagcatgagg accaggtgga gcagtataag 780aaggagctgg
agaagactta ttctgccaag ctggacaatg ccaggcagtc tgctgagagg
840aacagcaacc tggtgggggc tgcccacgag gagctgcagc agtcgcgcat
ccgcatcgac 900agcctctctg cccagctcag ccagctccag aagcagctgg
cagccaagga ggcgaagctt 960cgagacctgg aggactcact ggcccgtgag
cgggacacca gccggcggct gctggcggaa 1020aaggagcggg agatggccga
gatgcgggca aggatgcagc agcagctgga cgagtaccag 1080gagcttctgg
acatcaagct ggccctggac atggagatcc acgcctaccg caagctcttg
1140gagggcgagg aggagaggct acgcctgtcc cccagcccta cctcgcagcg
cagccgtggc 1200cgtgcttcct ctcactcatc ccagacacag ggtgggggca
gcgtcaccaa aaagcgcaaa 1260ctggagtcca ctgagagccg cagcagcttc
tcacagcacg cacgcactag cgggcgcgtg 1320gccgtggagg aggtggatga
ggagggcaag tttgtccggc tgcgcaacaa gtccaatgag 1380gaccagtcca
tgggcaattg gcagatcaag cgccagaatg gagatgatcc cttgctgact
1440taccggttcc caccaaagtt caccctgaag gctgggcagg tggtgacgat
ctgggctgca 1500ggagctgggg ccacccacag cccccctacc gacctggtgt
ggaaggcaca gaacacctgg 1560ggctgcggga acagcctgcg tacggctctc
atcaactcca ctggggaaga agtggccatg 1620cgcaagctgg tgcgctcagt
gactgtggtt gaggacgacg aggatgagga tggagatgac 1680ctgctccatc
accaccacgg ctcccactgc agcagctcgg gggaccccgc tgagtacaac
1740ctgcgctcgc gcaccgtgct gtgcgggacc tgcgggcagc ctgccgacaa
ggcatctgcc 1800agcggctcag gagcccaggt gggcggaccc atctcctctg
gctcttctgc ctccagtgtc 1860acggtcactc gcagctaccg cagtgtgggg
ggcagtgggg gtggcagctt cggggacaat 1920ctggtcaccc gctcctacct
cctgggcaac tccagccccc gaacccagag cccccagaac 1980tgcagcatca tgtaa
199520664PRTHomo sapiens 20Met Glu Thr Pro Ser Gln Arg Arg Ala Thr
Arg Ser Gly Ala Gln Ala 1 5 10 15 Ser Ser Thr Pro Leu Ser Pro Thr
Arg Ile Thr Arg Leu Gln Glu Lys 20 25 30 Glu Asp Leu Gln Glu Leu
Asn Asp Arg Leu Ala Val Tyr Ile Asp Arg 35 40 45 Val Arg Ser Leu
Glu Thr Glu Asn Ala Gly Leu Arg Leu Arg Ile Thr 50 55 60 Glu Ser
Glu Glu Val Val Ser Arg Glu Val Ser Gly Ile Lys Ala Ala 65 70 75 80
Tyr Glu Ala Glu Leu Gly Asp Ala Arg Lys Thr Leu Asp Ser Val Ala 85
90 95 Lys Glu Arg Ala Arg Leu Gln Leu Glu Leu Ser Lys Val Arg Glu
Glu 100 105 110 Phe Lys Glu Leu Lys Ala Arg Asn Thr Lys Lys Glu Gly
Asp Leu Ile 115 120 125 Ala Ala Gln Ala Arg Leu Lys Asp Leu Glu Ala
Leu Leu Asn Ser Lys 130 135 140 Glu Ala Ala Leu Ser Thr Ala Leu Ser
Glu Lys Arg Thr Leu Glu Gly 145 150 155 160 Glu Leu His Asp Leu Arg
Gly Gln Val Ala Lys Leu Glu Ala Ala Leu 165 170 175 Gly Glu Ala Lys
Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp 180 185 190 Ala Glu
Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe Gln Lys 195 200 205
Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg Arg His Glu Thr 210
215 220 Arg Leu Val Glu Ile Asp Asn Gly Lys Gln Arg Glu Phe Glu Ser
Arg 225 230 235 240 Leu Ala Asp Ala Leu Gln Glu Leu Arg Ala Gln His
Glu Asp Gln Val 245 250 255 Glu Gln Tyr Lys Lys Glu Leu Glu Lys Thr
Tyr Ser Ala Lys Leu Asp 260 265 270 Asn Ala Arg Gln Ser Ala Glu Arg
Asn Ser Asn Leu Val Gly Ala Ala 275 280 285 His Glu Glu Leu Gln Gln
Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala 290 295 300 Gln Leu Ser Gln
Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu 305 310 315 320 Arg
Asp Leu Glu Asp Ser Leu Ala Arg Glu Arg Asp Thr Ser Arg Arg 325 330
335 Leu Leu Ala Glu Lys Glu Arg Glu Met Ala Glu Met Arg Ala Arg Met
340 345 350 Gln Gln Gln Leu Asp Glu Tyr Gln Glu Leu Leu Asp Ile Lys
Leu Ala 355 360 365 Leu Asp Met Glu Ile His Ala Tyr Arg Lys Leu Leu
Glu Gly Glu Glu 370 375 380 Glu Arg Leu Arg Leu Ser Pro Ser Pro Thr
Ser Gln Arg Ser Arg Gly 385 390 395 400 Arg Ala Ser Ser His Ser Ser
Gln Thr Gln Gly Gly Gly Ser Val Thr 405 410 415 Lys Lys Arg Lys Leu
Glu Ser Thr Glu Ser Arg Ser Ser Phe Ser Gln 420 425 430 His Ala Arg
Thr Ser Gly Arg Val Ala Val Glu Glu Val Asp Glu Glu 435 440 445 Gly
Lys Phe Val Arg Leu Arg Asn Lys Ser Asn Glu Asp Gln Ser Met 450 455
460 Gly Asn Trp Gln Ile Lys Arg Gln Asn Gly Asp Asp Pro Leu Leu Thr
465 470 475 480 Tyr Arg Phe Pro Pro Lys Phe Thr Leu Lys Ala Gly Gln
Val Val Thr 485 490 495 Ile Trp Ala Ala Gly Ala Gly Ala Thr His Ser
Pro Pro Thr Asp Leu 500 505 510 Val Trp Lys Ala Gln Asn Thr Trp Gly
Cys Gly Asn Ser Leu Arg Thr 515 520 525 Ala Leu Ile Asn Ser Thr Gly
Glu Glu Val Ala Met Arg Lys Leu Val 530 535 540 Arg Ser Val Thr Val
Val Glu Asp Asp Glu Asp Glu Asp Gly Asp Asp 545 550 555 560 Leu Leu
His His His His Gly Ser His Cys Ser Ser Ser Gly Asp Pro 565 570 575
Ala Glu Tyr Asn Leu Arg Ser Arg Thr Val Leu Cys Gly Thr Cys Gly 580
585 590 Gln Pro Ala Asp Lys Ala Ser Ala Ser Gly Ser Gly Ala Gln Val
Gly 595 600 605 Gly Pro Ile Ser Ser Gly Ser Ser Ala Ser Ser Val Thr
Val Thr Arg 610 615 620 Ser Tyr Arg Ser Val Gly Gly Ser Gly Gly Gly
Ser Phe Gly Asp Asn 625 630 635 640 Leu Val Thr Arg Ser Tyr Leu Leu
Gly Asn Ser Ser Pro Arg Thr Gln 645 650 655 Ser Pro Gln Asn Cys Ser
Ile Met 660 211995DNAHomo sapiens 21atggagaccc cgtcccagcg
gcgcgccacc cgcagcgggg cgcaggccag ctccactccg 60ctgtcgccca cccgcatcac
ccggctgcag gagaaggagg acctgcagga gctcaatgat 120cgcttggcgg
tctacatcga ccgtgtgcgc tcgctggaaa cggagaacgc agggctgcgc
180cttcgcatca ccgagtctga agaggtggtc agccgcgagg tgtccggcat
caaggccgcc 240tacgaggccg agctcgggga tgcccgcaag acccttgact
cagtagccaa ggagcgcgcc 300cgcctgcagc tggagctgag caaagtgcgt
gaggagttta aggagctgaa agcgcgcaat 360accaagaagg agggtgacct
gatagctgct caggctcggc tgaaggacct ggaggctctg 420ctgaactcca
aggaggccgc actgagcact gctctcagtg agaagcgcac gctggagggc
480gagctgcatg atctgcgggg ccaggtggcc aagcttgagg cagccctagg
tgaggccaag 540aagcaacttc aggatgagat gctgcggcgg gtggatgctg
agaacaggct gcagaccatg 600aaggaggaac tggacttcca gaagaacatc
tacagtgagg agctgcgtga gaccaagcgc 660cgtcatgaga cccgactggt
ggagattgac aatgggaagc agcgtgagtt tgagagccgg 720ctggcggatg
cgctgcagga actgcgggcc cagcatgagg accaggtgga gcagtataag
780aaggagctgg agaagactta ttctgccaag ctggacaatg ccaggcagtc
tgctgagagg 840aacagcaacc tggtgggggc tgcccacgag gagctgcagc
agtcgcgcat ccgcatcgac 900agcctctctg cccagctcag ccagctccag
aagcagctgg cagccaagga ggcgaagctt 960cgagacctgg aggactcact
ggcccgtgag cgggacacca gccggcggct gctggcggaa 1020aaggagcggg
agatggccga gatgcgggca aggatgcagc agcagctgga cgagtaccag
1080gagcttctgg acatcaagct ggccctggac atggagatcc acgcctaccg
caagctcttg 1140gagggcgagg aggagaggct acgcctgtcc cccagcccta
cctcgcagcg cagccgtggc 1200cgtgcttcct ctcactcatc ccagacacag
ggtgggggca gcgtcaccaa aaagcgcaaa 1260ctggagtcca ctgagagccg
cagcagcttc tcacagcacg cacgcactag cgggcgcgtg 1320gccgtggagg
aggtggatga ggagggcaag tttgtccggc tgcgcaacaa gtccaatgag
1380gaccagtcca tgggcaattg gcagatcaag cgccagaatg gagatgatcc
cttgctgact 1440taccggttcc caccaaagtt caccctgaag gctgggcagg
tggtgacgat ctgggctgca 1500ggagctgggg ccacccacag cccccctacc
gacctggtgt ggaaggcaca gaacacctgg 1560ggctgcggga acagcctgcg
tacggctctc atcaactcca ctggggaaga agtggccatg 1620cgcaagctgg
tgcgctcagt gactgtggtt gaggacgacg aggatgagga tggagatgac
1680ctgctccatc accaccacgg ctcccactgc agcagctcgg gggaccccgc
tgagtacaac 1740ctgcgctcgc gcaccgtgct gtgcgggacc tgcgggcagc
ctgccgacaa ggcatctgcc 1800agcggctcag gagcccaggt gggcggaccc
atctcctctg gctcttctgc ctccagtgtc 1860acggtcactc gcagctaccg
cagtgtgggg ggcagtgggg gtggcagctt cggggacaat 1920ctggtcaccc
gctcctacct cctgggcaac tccagccccc gaacccagag cccccagaac
1980tgcagcatct tataa 199522664PRTHomo sapiens 22Met Glu Thr Pro Ser
Gln Arg Arg Ala Thr Arg Ser Gly Ala Gln Ala 1 5 10 15 Ser Ser Thr
Pro Leu Ser Pro Thr Arg Ile Thr Arg Leu Gln Glu Lys 20 25 30 Glu
Asp Leu Gln Glu Leu Asn Asp Arg Leu Ala Val Tyr Ile Asp Arg 35 40
45 Val Arg Ser Leu Glu Thr Glu Asn Ala Gly Leu Arg Leu Arg Ile Thr
50 55 60 Glu Ser Glu Glu Val Val Ser Arg Glu Val Ser Gly Ile Lys
Ala Ala 65 70 75 80 Tyr Glu Ala Glu Leu Gly Asp Ala Arg Lys Thr Leu
Asp Ser Val Ala 85 90 95 Lys Glu Arg Ala Arg Leu Gln Leu Glu Leu
Ser Lys Val Arg Glu Glu 100 105 110 Phe Lys Glu Leu Lys Ala Arg Asn
Thr Lys Lys Glu Gly Asp Leu Ile 115 120 125 Ala Ala Gln Ala Arg Leu
Lys Asp Leu Glu Ala Leu Leu Asn Ser Lys 130 135 140 Glu Ala Ala Leu
Ser Thr Ala Leu Ser Glu Lys Arg Thr Leu Glu Gly 145 150 155 160 Glu
Leu His Asp Leu Arg Gly Gln Val Ala Lys Leu Glu Ala Ala Leu 165 170
175 Gly Glu Ala Lys Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp
180 185 190 Ala Glu Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe
Gln Lys 195 200 205 Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg
Arg His Glu Thr 210 215 220 Arg Leu Val Glu Ile Asp Asn Gly Lys Gln
Arg Glu Phe Glu Ser Arg 225 230 235 240 Leu Ala Asp Ala Leu Gln Glu
Leu Arg Ala Gln His Glu Asp Gln Val 245 250 255 Glu Gln Tyr Lys Lys
Glu Leu Glu Lys Thr Tyr Ser Ala Lys Leu Asp 260 265 270 Asn Ala Arg
Gln Ser Ala Glu Arg Asn Ser Asn Leu Val Gly Ala Ala 275 280 285 His
Glu Glu Leu Gln Gln Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala 290 295
300 Gln Leu Ser Gln Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu
305 310 315 320 Arg Asp Leu Glu Asp Ser Leu Ala Arg Glu Arg Asp Thr
Ser Arg Arg 325 330 335 Leu Leu Ala Glu Lys Glu Arg Glu Met Ala Glu
Met Arg Ala Arg Met 340 345 350 Gln Gln Gln Leu Asp Glu Tyr Gln Glu
Leu Leu Asp Ile Lys Leu Ala 355 360 365 Leu Asp Met Glu Ile His Ala
Tyr Arg Lys Leu Leu Glu Gly Glu Glu 370 375 380 Glu Arg Leu Arg Leu
Ser Pro Ser Pro Thr Ser Gln Arg Ser Arg Gly 385 390 395 400 Arg Ala
Ser Ser His Ser Ser Gln Thr Gln Gly Gly Gly Ser Val Thr 405 410 415
Lys Lys Arg Lys Leu Glu Ser Thr Glu Ser Arg Ser Ser Phe Ser Gln 420
425 430 His Ala Arg Thr Ser Gly Arg Val Ala Val Glu Glu Val Asp Glu
Glu 435 440 445 Gly Lys Phe Val Arg Leu Arg Asn Lys Ser Asn Glu Asp
Gln Ser Met 450 455 460 Gly Asn Trp Gln Ile Lys Arg Gln Asn Gly Asp
Asp Pro Leu Leu Thr 465 470 475 480 Tyr Arg Phe Pro Pro Lys Phe Thr
Leu Lys Ala Gly Gln Val Val Thr 485 490 495 Ile Trp Ala Ala Gly Ala
Gly Ala Thr His Ser Pro Pro Thr Asp Leu 500 505 510 Val Trp Lys Ala
Gln Asn Thr Trp Gly Cys Gly Asn Ser Leu Arg Thr 515 520 525 Ala Leu
Ile Asn Ser Thr Gly Glu Glu Val Ala Met Arg Lys Leu Val 530 535 540
Arg Ser Val Thr Val Val Glu Asp Asp Glu Asp Glu Asp Gly Asp Asp 545
550 555 560 Leu Leu His His His His Gly Ser His Cys Ser Ser Ser Gly
Asp Pro 565 570 575 Ala Glu Tyr Asn Leu Arg Ser Arg Thr Val Leu Cys
Gly Thr Cys Gly 580 585 590 Gln Pro Ala Asp Lys Ala Ser Ala Ser Gly
Ser Gly Ala Gln Val Gly 595 600 605 Gly Pro Ile Ser Ser Gly Ser Ser
Ala Ser Ser Val Thr Val Thr Arg 610 615 620 Ser Tyr Arg Ser Val Gly
Gly Ser Gly Gly Gly Ser Phe Gly Asp Asn 625 630 635 640 Leu Val Thr
Arg Ser Tyr Leu Leu Gly Asn Ser Ser Pro Arg Thr Gln 645 650 655 Ser
Pro Gln Asn Cys Ser Ile Leu 660 231995DNAHomo sapiens 23atggagaccc
cgtcccagcg gcgcgccacc cgcagcgggg cgcaggccag ctccactccg 60ctgtcgccca
cccgcatcac ccggctgcag gagaaggagg
acctgcagga gctcaatgat 120cgcttggcgg tctacatcga ccgtgtgcgc
tcgctggaaa cggagaacgc agggctgcgc 180cttcgcatca ccgagtctga
agaggtggtc agccgcgagg tgtccggcat caaggccgcc 240tacgaggccg
agctcgggga tgcccgcaag acccttgact cagtagccaa ggagcgcgcc
300cgcctgcagc tggagctgag caaagtgcgt gaggagttta aggagctgaa
agcgcgcaat 360accaagaagg agggtgacct gatagctgct caggctcggc
tgaaggacct ggaggctctg 420ctgaactcca aggaggccgc actgagcact
gctctcagtg agaagcgcac gctggagggc 480gagctgcatg atctgcgggg
ccaggtggcc aagcttgagg cagccctagg tgaggccaag 540aagcaacttc
aggatgagat gctgcggcgg gtggatgctg agaacaggct gcagaccatg
600aaggaggaac tggacttcca gaagaacatc tacagtgagg agctgcgtga
gaccaagcgc 660cgtcatgaga cccgactggt ggagattgac aatgggaagc
agcgtgagtt tgagagccgg 720ctggcggatg cgctgcagga actgcgggcc
cagcatgagg accaggtgga gcagtataag 780aaggagctgg agaagactta
ttctgccaag ctggacaatg ccaggcagtc tgctgagagg 840aacagcaacc
tggtgggggc tgcccacgag gagctgcagc agtcgcgcat ccgcatcgac
900agcctctctg cccagctcag ccagctccag aagcagctgg cagccaagga
ggcgaagctt 960cgagacctgg aggactcact ggcccgtgag cgggacacca
gccggcggct gctggcggaa 1020aaggagcggg agatggccga gatgcgggca
aggatgcagc agcagctgga cgagtaccag 1080gagcttctgg acatcaagct
ggccctggac atggagatcc acgcctaccg caagctcttg 1140gagggcgagg
aggagaggct acgcctgtcc cccagcccta cctcgcagcg cagccgtggc
1200cgtgcttcct ctcactcatc ccagacacag ggtgggggca gcgtcaccaa
aaagcgcaaa 1260ctggagtcca ctgagagccg cagcagcttc tcacagcacg
cacgcactag cgggcgcgtg 1320gccgtggagg aggtggatga ggagggcaag
tttgtccggc tgcgcaacaa gtccaatgag 1380gaccagtcca tgggcaattg
gcagatcaag cgccagaatg gagatgatcc cttgctgact 1440taccggttcc
caccaaagtt caccctgaag gctgggcagg tggtgacgat ctgggctgca
1500ggagctgggg ccacccacag cccccctacc gacctggtgt ggaaggcaca
gaacacctgg 1560ggctgcggga acagcctgcg tacggctctc atcaactcca
ctggggaaga agtggccatg 1620cgcaagctgg tgcgctcagt gactgtggtt
gaggacgacg aggatgagga tggagatgac 1680ctgctccatc accaccacgg
ctcccactgc agcagctcgg gggaccccgc tgagtacaac 1740ctgcgctcgc
gcaccgtgct gtgcgggacc tgcgggcagc ctgccgacaa ggcatctgcc
1800agcggctcag gagcccaggt gggcggaccc atctcctctg gctcttctgc
ctccagtgtc 1860acggtcactc gcagctaccg cagtgtgggg ggcagtgggg
gtggcagctt cggggacaat 1920ctggtcaccc gctcctacct cctgggcaac
tccagccccc gaacccagag cccccagaac 1980tcaagcatca tgtaa
199524664PRTHomo sapiens 24Met Glu Thr Pro Ser Gln Arg Arg Ala Thr
Arg Ser Gly Ala Gln Ala 1 5 10 15 Ser Ser Thr Pro Leu Ser Pro Thr
Arg Ile Thr Arg Leu Gln Glu Lys 20 25 30 Glu Asp Leu Gln Glu Leu
Asn Asp Arg Leu Ala Val Tyr Ile Asp Arg 35 40 45 Val Arg Ser Leu
Glu Thr Glu Asn Ala Gly Leu Arg Leu Arg Ile Thr 50 55 60 Glu Ser
Glu Glu Val Val Ser Arg Glu Val Ser Gly Ile Lys Ala Ala 65 70 75 80
Tyr Glu Ala Glu Leu Gly Asp Ala Arg Lys Thr Leu Asp Ser Val Ala 85
90 95 Lys Glu Arg Ala Arg Leu Gln Leu Glu Leu Ser Lys Val Arg Glu
Glu 100 105 110 Phe Lys Glu Leu Lys Ala Arg Asn Thr Lys Lys Glu Gly
Asp Leu Ile 115 120 125 Ala Ala Gln Ala Arg Leu Lys Asp Leu Glu Ala
Leu Leu Asn Ser Lys 130 135 140 Glu Ala Ala Leu Ser Thr Ala Leu Ser
Glu Lys Arg Thr Leu Glu Gly 145 150 155 160 Glu Leu His Asp Leu Arg
Gly Gln Val Ala Lys Leu Glu Ala Ala Leu 165 170 175 Gly Glu Ala Lys
Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp 180 185 190 Ala Glu
Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe Gln Lys 195 200 205
Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg Arg His Glu Thr 210
215 220 Arg Leu Val Glu Ile Asp Asn Gly Lys Gln Arg Glu Phe Glu Ser
Arg 225 230 235 240 Leu Ala Asp Ala Leu Gln Glu Leu Arg Ala Gln His
Glu Asp Gln Val 245 250 255 Glu Gln Tyr Lys Lys Glu Leu Glu Lys Thr
Tyr Ser Ala Lys Leu Asp 260 265 270 Asn Ala Arg Gln Ser Ala Glu Arg
Asn Ser Asn Leu Val Gly Ala Ala 275 280 285 His Glu Glu Leu Gln Gln
Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala 290 295 300 Gln Leu Ser Gln
Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu 305 310 315 320 Arg
Asp Leu Glu Asp Ser Leu Ala Arg Glu Arg Asp Thr Ser Arg Arg 325 330
335 Leu Leu Ala Glu Lys Glu Arg Glu Met Ala Glu Met Arg Ala Arg Met
340 345 350 Gln Gln Gln Leu Asp Glu Tyr Gln Glu Leu Leu Asp Ile Lys
Leu Ala 355 360 365 Leu Asp Met Glu Ile His Ala Tyr Arg Lys Leu Leu
Glu Gly Glu Glu 370 375 380 Glu Arg Leu Arg Leu Ser Pro Ser Pro Thr
Ser Gln Arg Ser Arg Gly 385 390 395 400 Arg Ala Ser Ser His Ser Ser
Gln Thr Gln Gly Gly Gly Ser Val Thr 405 410 415 Lys Lys Arg Lys Leu
Glu Ser Thr Glu Ser Arg Ser Ser Phe Ser Gln 420 425 430 His Ala Arg
Thr Ser Gly Arg Val Ala Val Glu Glu Val Asp Glu Glu 435 440 445 Gly
Lys Phe Val Arg Leu Arg Asn Lys Ser Asn Glu Asp Gln Ser Met 450 455
460 Gly Asn Trp Gln Ile Lys Arg Gln Asn Gly Asp Asp Pro Leu Leu Thr
465 470 475 480 Tyr Arg Phe Pro Pro Lys Phe Thr Leu Lys Ala Gly Gln
Val Val Thr 485 490 495 Ile Trp Ala Ala Gly Ala Gly Ala Thr His Ser
Pro Pro Thr Asp Leu 500 505 510 Val Trp Lys Ala Gln Asn Thr Trp Gly
Cys Gly Asn Ser Leu Arg Thr 515 520 525 Ala Leu Ile Asn Ser Thr Gly
Glu Glu Val Ala Met Arg Lys Leu Val 530 535 540 Arg Ser Val Thr Val
Val Glu Asp Asp Glu Asp Glu Asp Gly Asp Asp 545 550 555 560 Leu Leu
His His His His Gly Ser His Cys Ser Ser Ser Gly Asp Pro 565 570 575
Ala Glu Tyr Asn Leu Arg Ser Arg Thr Val Leu Cys Gly Thr Cys Gly 580
585 590 Gln Pro Ala Asp Lys Ala Ser Ala Ser Gly Ser Gly Ala Gln Val
Gly 595 600 605 Gly Pro Ile Ser Ser Gly Ser Ser Ala Ser Ser Val Thr
Val Thr Arg 610 615 620 Ser Tyr Arg Ser Val Gly Gly Ser Gly Gly Gly
Ser Phe Gly Asp Asn 625 630 635 640 Leu Val Thr Arg Ser Tyr Leu Leu
Gly Asn Ser Ser Pro Arg Thr Gln 645 650 655 Ser Pro Gln Asn Ser Ser
Ile Met 660 251845DNAHomo sapiens 25atggagaccc cgtcccagcg
gcgcgccacc cgcagcgggg cgcaggccag ctccactccg 60ctgtcgccca cccgcatcac
ccggctgcag gagaaggagg acctgcagga gctcaatgat 120cgcttggcgg
tctacatcga ccgtgtgcgc tcgctggaaa cggagaacgc agggctgcgc
180cttcgcatca ccgagtctga agaggtggtc agccgcgagg tgtccggcat
caaggccgcc 240tacgaggccg agctcgggga tgcccgcaag acccttgact
cagtagccaa ggagcgcgcc 300cgcctgcagc tggagctgag caaagtgcgt
gaggagttta aggagctgaa agcgcgcaat 360accaagaagg agggtgacct
gatagctgct caggctcggc tgaaggacct ggaggctctg 420ctgaactcca
aggaggccgc actgagcact gctctcagtg agaagcgcac gctggagggc
480gagctgcatg atctgcgggg ccaggtggcc aagcttgagg cagccctagg
tgaggccaag 540aagcaacttc aggatgagat gctgcggcgg gtggatgctg
agaacaggct gcagaccatg 600aaggaggaac tggacttcca gaagaacatc
tacagtgagg agctgcgtga gaccaagcgc 660cgtcatgaga cccgactggt
ggagattgac aatgggaagc agcgtgagtt tgagagccgg 720ctggcggatg
cgctgcagga actgcgggcc cagcatgagg accaggtgga gcagtataag
780aaggagctgg agaagactta ttctgccaag ctggacaatg ccaggcagtc
tgctgagagg 840aacagcaacc tggtgggggc tgcccacgag gagctgcagc
agtcgcgcat ccgcatcgac 900agcctctctg cccagctcag ccagctccag
aagcagctgg cagccaagga ggcgaagctt 960cgagacctgg aggactcact
ggcccgtgag cgggacacca gccggcggct gctggcggaa 1020aaggagcggg
agatggccga gatgcgggca aggatgcagc agcagctgga cgagtaccag
1080gagcttctgg acatcaagct ggccctggac atggagatcc acgcctaccg
caagctcttg 1140gagggcgagg aggagaggct acgcctgtcc cccagcccta
cctcgcagcg cagccgtggc 1200cgtgcttcct ctcactcatc ccagacacag
ggtgggggca gcgtcaccaa aaagcgcaaa 1260ctggagtcca ctgagagccg
cagcagcttc tcacagcacg cacgcactag cgggcgcgtg 1320gccgtggagg
aggtggatga ggagggcaag tttgtccggc tgcgcaacaa gtccaatgag
1380gaccagtcca tgggcaattg gcagatcaag cgccagaatg gagatgatcc
cttgctgact 1440taccggttcc caccaaagtt caccctgaag gctgggcagg
tggtgacgat ctgggctgca 1500ggagctgggg ccacccacag cccccctacc
gacctggtgt ggaaggcaca gaacacctgg 1560ggctgcggga acagcctgcg
tacggctctc atcaactcca ctggggaaga agtggccatg 1620cgcaagctgg
tgcgctcagt gactgtggtt gaggacgacg aggatgagga tggagatgac
1680ctgctccatc accaccacgg ctcccactgc agcagctcgg gggaccccgc
tgagtacaac 1740ctgcgctcgc gcaccgtgct gtgcgggacc tgcgggcagc
ctgccgacaa ggcatctgcc 1800agcggctcag gagcccagag cccccagaac
tgcagcatca tgtaa 184526614PRTHomo sapiens 26Met Glu Thr Pro Ser Gln
Arg Arg Ala Thr Arg Ser Gly Ala Gln Ala 1 5 10 15 Ser Ser Thr Pro
Leu Ser Pro Thr Arg Ile Thr Arg Leu Gln Glu Lys 20 25 30 Glu Asp
Leu Gln Glu Leu Asn Asp Arg Leu Ala Val Tyr Ile Asp Arg 35 40 45
Val Arg Ser Leu Glu Thr Glu Asn Ala Gly Leu Arg Leu Arg Ile Thr 50
55 60 Glu Ser Glu Glu Val Val Ser Arg Glu Val Ser Gly Ile Lys Ala
Ala 65 70 75 80 Tyr Glu Ala Glu Leu Gly Asp Ala Arg Lys Thr Leu Asp
Ser Val Ala 85 90 95 Lys Glu Arg Ala Arg Leu Gln Leu Glu Leu Ser
Lys Val Arg Glu Glu 100 105 110 Phe Lys Glu Leu Lys Ala Arg Asn Thr
Lys Lys Glu Gly Asp Leu Ile 115 120 125 Ala Ala Gln Ala Arg Leu Lys
Asp Leu Glu Ala Leu Leu Asn Ser Lys 130 135 140 Glu Ala Ala Leu Ser
Thr Ala Leu Ser Glu Lys Arg Thr Leu Glu Gly 145 150 155 160 Glu Leu
His Asp Leu Arg Gly Gln Val Ala Lys Leu Glu Ala Ala Leu 165 170 175
Gly Glu Ala Lys Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp 180
185 190 Ala Glu Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe Gln
Lys 195 200 205 Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg Arg
His Glu Thr 210 215 220 Arg Leu Val Glu Ile Asp Asn Gly Lys Gln Arg
Glu Phe Glu Ser Arg 225 230 235 240 Leu Ala Asp Ala Leu Gln Glu Leu
Arg Ala Gln His Glu Asp Gln Val 245 250 255 Glu Gln Tyr Lys Lys Glu
Leu Glu Lys Thr Tyr Ser Ala Lys Leu Asp 260 265 270 Asn Ala Arg Gln
Ser Ala Glu Arg Asn Ser Asn Leu Val Gly Ala Ala 275 280 285 His Glu
Glu Leu Gln Gln Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala 290 295 300
Gln Leu Ser Gln Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu 305
310 315 320 Arg Asp Leu Glu Asp Ser Leu Ala Arg Glu Arg Asp Thr Ser
Arg Arg 325 330 335 Leu Leu Ala Glu Lys Glu Arg Glu Met Ala Glu Met
Arg Ala Arg Met 340 345 350 Gln Gln Gln Leu Asp Glu Tyr Gln Glu Leu
Leu Asp Ile Lys Leu Ala 355 360 365 Leu Asp Met Glu Ile His Ala Tyr
Arg Lys Leu Leu Glu Gly Glu Glu 370 375 380 Glu Arg Leu Arg Leu Ser
Pro Ser Pro Thr Ser Gln Arg Ser Arg Gly 385 390 395 400 Arg Ala Ser
Ser His Ser Ser Gln Thr Gln Gly Gly Gly Ser Val Thr 405 410 415 Lys
Lys Arg Lys Leu Glu Ser Thr Glu Ser Arg Ser Ser Phe Ser Gln 420 425
430 His Ala Arg Thr Ser Gly Arg Val Ala Val Glu Glu Val Asp Glu Glu
435 440 445 Gly Lys Phe Val Arg Leu Arg Asn Lys Ser Asn Glu Asp Gln
Ser Met 450 455 460 Gly Asn Trp Gln Ile Lys Arg Gln Asn Gly Asp Asp
Pro Leu Leu Thr 465 470 475 480 Tyr Arg Phe Pro Pro Lys Phe Thr Leu
Lys Ala Gly Gln Val Val Thr 485 490 495 Ile Trp Ala Ala Gly Ala Gly
Ala Thr His Ser Pro Pro Thr Asp Leu 500 505 510 Val Trp Lys Ala Gln
Asn Thr Trp Gly Cys Gly Asn Ser Leu Arg Thr 515 520 525 Ala Leu Ile
Asn Ser Thr Gly Glu Glu Val Ala Met Arg Lys Leu Val 530 535 540 Arg
Ser Val Thr Val Val Glu Asp Asp Glu Asp Glu Asp Gly Asp Asp 545 550
555 560 Leu Leu His His His His Gly Ser His Cys Ser Ser Ser Gly Asp
Pro 565 570 575 Ala Glu Tyr Asn Leu Arg Ser Arg Thr Val Leu Cys Gly
Thr Cys Gly 580 585 590 Gln Pro Ala Asp Lys Ala Ser Ala Ser Gly Ser
Gly Ala Gln Ser Pro 595 600 605 Gln Asn Cys Ser Ile Met 610
271845DNAHomo sapiens 27atggagaccc cgtcccagcg gcgcgccacc cgcagcgggg
cgcaggccag ctccactccg 60ctgtcgccca cccgcatcac ccggctgcag gagaaggagg
acctgcagga gctcaatgat 120cgcttggcgg tctacatcga ccgtgtgcgc
tcgctggaaa cggagaacgc agggctgcgc 180cttcgcatca ccgagtctga
agaggtggtc agccgcgagg tgtccggcat caaggccgcc 240tacgaggccg
agctcgggga tgcccgcaag acccttgact cagtagccaa ggagcgcgcc
300cgcctgcagc tggagctgag caaagtgcgt gaggagttta aggagctgaa
agcgcgcaat 360accaagaagg agggtgacct gatagctgct caggctcggc
tgaaggacct ggaggctctg 420ctgaactcca aggaggccgc actgagcact
gctctcagtg agaagcgcac gctggagggc 480gagctgcatg atctgcgggg
ccaggtggcc aagcttgagg cagccctagg tgaggccaag 540aagcaacttc
aggatgagat gctgcggcgg gtggatgctg agaacaggct gcagaccatg
600aaggaggaac tggacttcca gaagaacatc tacagtgagg agctgcgtga
gaccaagcgc 660cgtcatgaga cccgactggt ggagattgac aatgggaagc
agcgtgagtt tgagagccgg 720ctggcggatg cgctgcagga actgcgggcc
cagcatgagg accaggtgga gcagtataag 780aaggagctgg agaagactta
ttctgccaag ctggacaatg ccaggcagtc tgctgagagg 840aacagcaacc
tggtgggggc tgcccacgag gagctgcagc agtcgcgcat ccgcatcgac
900agcctctctg cccagctcag ccagctccag aagcagctgg cagccaagga
ggcgaagctt 960cgagacctgg aggactcact ggcccgtgag cgggacacca
gccggcggct gctggcggaa 1020aaggagcggg agatggccga gatgcgggca
aggatgcagc agcagctgga cgagtaccag 1080gagcttctgg acatcaagct
ggccctggac atggagatcc acgcctaccg caagctcttg 1140gagggcgagg
aggagaggct acgcctgtcc cccagcccta cctcgcagcg cagccgtggc
1200cgtgcttcct ctcactcatc ccagacacag ggtgggggca gcgtcaccaa
aaagcgcaaa 1260ctggagtcca ctgagagccg cagcagcttc tcacagcacg
cacgcactag cgggcgcgtg 1320gccgtggagg aggtggatga ggagggcaag
tttgtccggc tgcgcaacaa gtccaatgag 1380gaccagtcca tgggcaattg
gcagatcaag cgccagaatg gagatgatcc cttgctgact 1440taccggttcc
caccaaagtt caccctgaag gctgggcagg tggtgacgat ctgggctgca
1500ggagctgggg ccacccacag cccccctacc gacctggtgt ggaaggcaca
gaacacctgg 1560ggctgcggga acagcctgcg tacggctctc atcaactcca
ctggggaaga agtggccatg 1620cgcaagctgg tgcgctcagt gactgtggtt
gaggacgacg aggatgagga tggagatgac 1680ctgctccatc accaccacgg
ctcccactgc agcagctcgg gggaccccgc tgagtacaac 1740ctgcgctcgc
gcaccgtgct gtgcgggacc tgcgggcagc ctgccgacaa ggcatctgcc
1800agcggctcag gagcccagag cccccagaac tgcagcatct tataa
184528614PRTHomo sapiens 28Met Glu Thr Pro Ser Gln Arg Arg Ala Thr
Arg Ser Gly Ala Gln Ala 1 5 10 15 Ser Ser Thr Pro Leu Ser Pro Thr
Arg Ile Thr Arg Leu Gln Glu Lys 20 25 30 Glu Asp Leu Gln Glu Leu
Asn Asp Arg Leu Ala Val Tyr Ile Asp Arg 35 40 45 Val Arg Ser Leu
Glu Thr Glu Asn Ala Gly Leu Arg Leu Arg Ile Thr 50 55 60 Glu Ser
Glu Glu Val Val Ser Arg Glu Val Ser Gly Ile Lys Ala Ala 65 70 75 80
Tyr Glu Ala Glu Leu Gly Asp Ala Arg Lys Thr Leu Asp Ser Val Ala 85
90 95 Lys Glu Arg Ala Arg Leu Gln Leu Glu Leu Ser Lys Val Arg Glu
Glu 100 105 110 Phe Lys Glu Leu Lys Ala Arg Asn Thr Lys Lys Glu Gly
Asp Leu Ile 115 120 125 Ala Ala Gln Ala Arg Leu Lys Asp Leu Glu Ala
Leu Leu Asn Ser Lys 130 135 140 Glu Ala Ala Leu
Ser Thr Ala Leu Ser Glu Lys Arg Thr Leu Glu Gly 145 150 155 160 Glu
Leu His Asp Leu Arg Gly Gln Val Ala Lys Leu Glu Ala Ala Leu 165 170
175 Gly Glu Ala Lys Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp
180 185 190 Ala Glu Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe
Gln Lys 195 200 205 Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg
Arg His Glu Thr 210 215 220 Arg Leu Val Glu Ile Asp Asn Gly Lys Gln
Arg Glu Phe Glu Ser Arg 225 230 235 240 Leu Ala Asp Ala Leu Gln Glu
Leu Arg Ala Gln His Glu Asp Gln Val 245 250 255 Glu Gln Tyr Lys Lys
Glu Leu Glu Lys Thr Tyr Ser Ala Lys Leu Asp 260 265 270 Asn Ala Arg
Gln Ser Ala Glu Arg Asn Ser Asn Leu Val Gly Ala Ala 275 280 285 His
Glu Glu Leu Gln Gln Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala 290 295
300 Gln Leu Ser Gln Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu
305 310 315 320 Arg Asp Leu Glu Asp Ser Leu Ala Arg Glu Arg Asp Thr
Ser Arg Arg 325 330 335 Leu Leu Ala Glu Lys Glu Arg Glu Met Ala Glu
Met Arg Ala Arg Met 340 345 350 Gln Gln Gln Leu Asp Glu Tyr Gln Glu
Leu Leu Asp Ile Lys Leu Ala 355 360 365 Leu Asp Met Glu Ile His Ala
Tyr Arg Lys Leu Leu Glu Gly Glu Glu 370 375 380 Glu Arg Leu Arg Leu
Ser Pro Ser Pro Thr Ser Gln Arg Ser Arg Gly 385 390 395 400 Arg Ala
Ser Ser His Ser Ser Gln Thr Gln Gly Gly Gly Ser Val Thr 405 410 415
Lys Lys Arg Lys Leu Glu Ser Thr Glu Ser Arg Ser Ser Phe Ser Gln 420
425 430 His Ala Arg Thr Ser Gly Arg Val Ala Val Glu Glu Val Asp Glu
Glu 435 440 445 Gly Lys Phe Val Arg Leu Arg Asn Lys Ser Asn Glu Asp
Gln Ser Met 450 455 460 Gly Asn Trp Gln Ile Lys Arg Gln Asn Gly Asp
Asp Pro Leu Leu Thr 465 470 475 480 Tyr Arg Phe Pro Pro Lys Phe Thr
Leu Lys Ala Gly Gln Val Val Thr 485 490 495 Ile Trp Ala Ala Gly Ala
Gly Ala Thr His Ser Pro Pro Thr Asp Leu 500 505 510 Val Trp Lys Ala
Gln Asn Thr Trp Gly Cys Gly Asn Ser Leu Arg Thr 515 520 525 Ala Leu
Ile Asn Ser Thr Gly Glu Glu Val Ala Met Arg Lys Leu Val 530 535 540
Arg Ser Val Thr Val Val Glu Asp Asp Glu Asp Glu Asp Gly Asp Asp 545
550 555 560 Leu Leu His His His His Gly Ser His Cys Ser Ser Ser Gly
Asp Pro 565 570 575 Ala Glu Tyr Asn Leu Arg Ser Arg Thr Val Leu Cys
Gly Thr Cys Gly 580 585 590 Gln Pro Ala Asp Lys Ala Ser Ala Ser Gly
Ser Gly Ala Gln Ser Pro 595 600 605 Gln Asn Cys Ser Ile Leu 610
291845DNAHomo sapiens 29atggagaccc cgtcccagcg gcgcgccacc cgcagcgggg
cgcaggccag ctccactccg 60ctgtcgccca cccgcatcac ccggctgcag gagaaggagg
acctgcagga gctcaatgat 120cgcttggcgg tctacatcga ccgtgtgcgc
tcgctggaaa cggagaacgc agggctgcgc 180cttcgcatca ccgagtctga
agaggtggtc agccgcgagg tgtccggcat caaggccgcc 240tacgaggccg
agctcgggga tgcccgcaag acccttgact cagtagccaa ggagcgcgcc
300cgcctgcagc tggagctgag caaagtgcgt gaggagttta aggagctgaa
agcgcgcaat 360accaagaagg agggtgacct gatagctgct caggctcggc
tgaaggacct ggaggctctg 420ctgaactcca aggaggccgc actgagcact
gctctcagtg agaagcgcac gctggagggc 480gagctgcatg atctgcgggg
ccaggtggcc aagcttgagg cagccctagg tgaggccaag 540aagcaacttc
aggatgagat gctgcggcgg gtggatgctg agaacaggct gcagaccatg
600aaggaggaac tggacttcca gaagaacatc tacagtgagg agctgcgtga
gaccaagcgc 660cgtcatgaga cccgactggt ggagattgac aatgggaagc
agcgtgagtt tgagagccgg 720ctggcggatg cgctgcagga actgcgggcc
cagcatgagg accaggtgga gcagtataag 780aaggagctgg agaagactta
ttctgccaag ctggacaatg ccaggcagtc tgctgagagg 840aacagcaacc
tggtgggggc tgcccacgag gagctgcagc agtcgcgcat ccgcatcgac
900agcctctctg cccagctcag ccagctccag aagcagctgg cagccaagga
ggcgaagctt 960cgagacctgg aggactcact ggcccgtgag cgggacacca
gccggcggct gctggcggaa 1020aaggagcggg agatggccga gatgcgggca
aggatgcagc agcagctgga cgagtaccag 1080gagcttctgg acatcaagct
ggccctggac atggagatcc acgcctaccg caagctcttg 1140gagggcgagg
aggagaggct acgcctgtcc cccagcccta cctcgcagcg cagccgtggc
1200cgtgcttcct ctcactcatc ccagacacag ggtgggggca gcgtcaccaa
aaagcgcaaa 1260ctggagtcca ctgagagccg cagcagcttc tcacagcacg
cacgcactag cgggcgcgtg 1320gccgtggagg aggtggatga ggagggcaag
tttgtccggc tgcgcaacaa gtccaatgag 1380gaccagtcca tgggcaattg
gcagatcaag cgccagaatg gagatgatcc cttgctgact 1440taccggttcc
caccaaagtt caccctgaag gctgggcagg tggtgacgat ctgggctgca
1500ggagctgggg ccacccacag cccccctacc gacctggtgt ggaaggcaca
gaacacctgg 1560ggctgcggga acagcctgcg tacggctctc atcaactcca
ctggggaaga agtggccatg 1620cgcaagctgg tgcgctcagt gactgtggtt
gaggacgacg aggatgagga tggagatgac 1680ctgctccatc accaccacgg
ctcccactgc agcagctcgg gggaccccgc tgagtacaac 1740ctgcgctcgc
gcaccgtgct gtgcgggacc tgcgggcagc ctgccgacaa ggcatctgcc
1800agcggctcag gagcccagag cccccagaac tcaagcatca tgtaa
184530614PRTHomo sapiens 30Met Glu Thr Pro Ser Gln Arg Arg Ala Thr
Arg Ser Gly Ala Gln Ala 1 5 10 15 Ser Ser Thr Pro Leu Ser Pro Thr
Arg Ile Thr Arg Leu Gln Glu Lys 20 25 30 Glu Asp Leu Gln Glu Leu
Asn Asp Arg Leu Ala Val Tyr Ile Asp Arg 35 40 45 Val Arg Ser Leu
Glu Thr Glu Asn Ala Gly Leu Arg Leu Arg Ile Thr 50 55 60 Glu Ser
Glu Glu Val Val Ser Arg Glu Val Ser Gly Ile Lys Ala Ala 65 70 75 80
Tyr Glu Ala Glu Leu Gly Asp Ala Arg Lys Thr Leu Asp Ser Val Ala 85
90 95 Lys Glu Arg Ala Arg Leu Gln Leu Glu Leu Ser Lys Val Arg Glu
Glu 100 105 110 Phe Lys Glu Leu Lys Ala Arg Asn Thr Lys Lys Glu Gly
Asp Leu Ile 115 120 125 Ala Ala Gln Ala Arg Leu Lys Asp Leu Glu Ala
Leu Leu Asn Ser Lys 130 135 140 Glu Ala Ala Leu Ser Thr Ala Leu Ser
Glu Lys Arg Thr Leu Glu Gly 145 150 155 160 Glu Leu His Asp Leu Arg
Gly Gln Val Ala Lys Leu Glu Ala Ala Leu 165 170 175 Gly Glu Ala Lys
Lys Gln Leu Gln Asp Glu Met Leu Arg Arg Val Asp 180 185 190 Ala Glu
Asn Arg Leu Gln Thr Met Lys Glu Glu Leu Asp Phe Gln Lys 195 200 205
Asn Ile Tyr Ser Glu Glu Leu Arg Glu Thr Lys Arg Arg His Glu Thr 210
215 220 Arg Leu Val Glu Ile Asp Asn Gly Lys Gln Arg Glu Phe Glu Ser
Arg 225 230 235 240 Leu Ala Asp Ala Leu Gln Glu Leu Arg Ala Gln His
Glu Asp Gln Val 245 250 255 Glu Gln Tyr Lys Lys Glu Leu Glu Lys Thr
Tyr Ser Ala Lys Leu Asp 260 265 270 Asn Ala Arg Gln Ser Ala Glu Arg
Asn Ser Asn Leu Val Gly Ala Ala 275 280 285 His Glu Glu Leu Gln Gln
Ser Arg Ile Arg Ile Asp Ser Leu Ser Ala 290 295 300 Gln Leu Ser Gln
Leu Gln Lys Gln Leu Ala Ala Lys Glu Ala Lys Leu 305 310 315 320 Arg
Asp Leu Glu Asp Ser Leu Ala Arg Glu Arg Asp Thr Ser Arg Arg 325 330
335 Leu Leu Ala Glu Lys Glu Arg Glu Met Ala Glu Met Arg Ala Arg Met
340 345 350 Gln Gln Gln Leu Asp Glu Tyr Gln Glu Leu Leu Asp Ile Lys
Leu Ala 355 360 365 Leu Asp Met Glu Ile His Ala Tyr Arg Lys Leu Leu
Glu Gly Glu Glu 370 375 380 Glu Arg Leu Arg Leu Ser Pro Ser Pro Thr
Ser Gln Arg Ser Arg Gly 385 390 395 400 Arg Ala Ser Ser His Ser Ser
Gln Thr Gln Gly Gly Gly Ser Val Thr 405 410 415 Lys Lys Arg Lys Leu
Glu Ser Thr Glu Ser Arg Ser Ser Phe Ser Gln 420 425 430 His Ala Arg
Thr Ser Gly Arg Val Ala Val Glu Glu Val Asp Glu Glu 435 440 445 Gly
Lys Phe Val Arg Leu Arg Asn Lys Ser Asn Glu Asp Gln Ser Met 450 455
460 Gly Asn Trp Gln Ile Lys Arg Gln Asn Gly Asp Asp Pro Leu Leu Thr
465 470 475 480 Tyr Arg Phe Pro Pro Lys Phe Thr Leu Lys Ala Gly Gln
Val Val Thr 485 490 495 Ile Trp Ala Ala Gly Ala Gly Ala Thr His Ser
Pro Pro Thr Asp Leu 500 505 510 Val Trp Lys Ala Gln Asn Thr Trp Gly
Cys Gly Asn Ser Leu Arg Thr 515 520 525 Ala Leu Ile Asn Ser Thr Gly
Glu Glu Val Ala Met Arg Lys Leu Val 530 535 540 Arg Ser Val Thr Val
Val Glu Asp Asp Glu Asp Glu Asp Gly Asp Asp 545 550 555 560 Leu Leu
His His His His Gly Ser His Cys Ser Ser Ser Gly Asp Pro 565 570 575
Ala Glu Tyr Asn Leu Arg Ser Arg Thr Val Leu Cys Gly Thr Cys Gly 580
585 590 Gln Pro Ala Asp Lys Ala Ser Ala Ser Gly Ser Gly Ala Gln Ser
Pro 595 600 605 Gln Asn Ser Ser Ile Met 610 314PRTArtificial
sequenceSynthetic Peptide 31Cys Xaa Xaa Xaa 1 324PRTArtificial
sequenceSynthetic Peptide 32Cys Ser Ile Met 1 334PRTArtificial
sequenceSynthetic Peptide 33Ser Ser Ile Met 1 344PRTArtificial
sequenceSynthetic Peptide 34Cys Ser Ile Leu 1
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