U.S. patent application number 13/393141 was filed with the patent office on 2012-11-08 for sdf-1 delivery for treating ischemic tissue.
Invention is credited to Rahul Aras, Timothy R. Miller, Joseph Pastore, Marc S. Penn.
Application Number | 20120283315 13/393141 |
Document ID | / |
Family ID | 43628693 |
Filed Date | 2012-11-08 |
United States Patent
Application |
20120283315 |
Kind Code |
A1 |
Penn; Marc S. ; et
al. |
November 8, 2012 |
SDF-1 DELIVERY FOR TREATING ISCHEMIC TISSUE
Abstract
A method of treating a cardiomyopathy in a subject includes
administering directly to or expressing locally in a weakened,
ischemic, and/or peri-infarct region of myocardial tissue of the
subject an amount of SDF-1 effective to cause functional
improvement in at least one of the following parameters: left
ventricular volume, left ventricular area, left ventricular
dimension, cardiac function, 6-minute walk test, or New York Heart
Association (NYHA) functional classification.
Inventors: |
Penn; Marc S.; (Beachwood,
OH) ; Aras; Rahul; (Braodview Heights, OH) ;
Pastore; Joseph; (Mentor, OH) ; Miller; Timothy
R.; (Cleveland Heights, OH) |
Family ID: |
43628693 |
Appl. No.: |
13/393141 |
Filed: |
August 30, 2010 |
PCT Filed: |
August 30, 2010 |
PCT NO: |
PCT/US2010/047175 |
371 Date: |
June 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61237775 |
Aug 28, 2009 |
|
|
|
61334216 |
May 13, 2010 |
|
|
|
Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K 48/005 20130101;
A61P 9/00 20180101; C07K 2319/02 20130101; A61K 38/19 20130101;
C07K 2319/09 20130101; A61K 9/0019 20130101; A61K 31/711 20130101;
A61K 38/00 20130101; A61P 9/10 20180101; C07K 14/521 20130101 |
Class at
Publication: |
514/44.R |
International
Class: |
A61K 31/711 20060101
A61K031/711; A61P 9/10 20060101 A61P009/10 |
Claims
1.-40. (canceled)
41. A method of treating a myocardial infarction in a large mammal
comprising: administering SDF-1 plasmid to the peri-infarct region
of the mammal by catheterization, SDF-1 being expressed from the
peri-infarct region at an amount effective to cause functional
improvement in at least one of the following parameters: left
ventricular volume, left ventricular area, left ventricular
dimension, cardiac function, 6-minute walk test, or New York Heart
Association (NYHA) functional classification.
42. The method of claim 41, the amount of SDF-1 administered to the
weakened, ischemic, and/or peri-infarct region is effective to
cause functional improvement in left ventricular end systolic
volume, left ventricular ejection fraction, wall motion score
index, left ventricular end diastolic length, left ventricular end
systolic length, left ventricular end diastolic area, left
ventricular end systolic area, or left ventricular end diastolic
volume.
43. The method of claim 42, wherein the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to improve left ventricular end systolic volume.
44. The method of claim 42, wherein the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to improve left ventricular ejection fraction.
45. The method of claim 43, wherein the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to improve left ventricular end systolic volume by at
least about 10%.
46. The method of claim 44, wherein the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to improve left ventricular ejection fraction by at
least about 10%.
47. The method of claim 41, wherein the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to improve 6 minute walk distance by at least about 30
meters or improve NYHA class by at least 1 class.
48. The method of claim 41, wherein the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to improve left ventricular end systolic volume by at
least about 10%, improve left ventricular ejection fraction by at
least about 10%, improve wall motion score index by about 5%,
improve 6 minute walk distance by at least about 30 meters, and
improve NYHA class by at least 1 class.
49. The method of claim 41, wherein the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to substantially improve vasculogenesis of the
weakened, ischemic, and/or peri-infarct region.
50. The method of claim 49, wherein the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to increase vasculogenesis by at least 20% based on
vessel density or measured by myocardial perfusion imaging with an
improvement in summed rest score, summed stress score, and/or
summed difference score of at least about 10%.
51. The method of claim 41, wherein the SDF-1 is administered by
injecting a solution comprising SDF-1 expressing plasmid DNA in the
weakened, ischemic, and/or peri-infarct region and expressing SDF-1
from the weakened, ischemic, and/or peri-infarct region.
52. The method of claim 51, wherein the SDF-1 is expressed from the
weakened, ischemic, and/or peri-infarct region at an amount
effective to improve left ventricular end systolic volume.
53. The method of claim 51, wherein the SDF-1 plasmid is
administered to the weakened, ischemic, and/or peri-infarct region
in multiple injections of the solution with each injection
comprising about 0.33 mg/ml to about 5 mg/ml of SDF-1
plasmid/solution.
54. The method of claim 53, wherein each injection has a volume of
at least about 0.2 ml.
55. The method of claim 54, wherein the SDF-1 plasmid is
administered to the weakened, ischemic, and/or peri-infarct region
in at least about 10 injections.
56. The method of claim 55, wherein the amount of SDF-1 plasmid
administered to the weakened, ischemic, and/or peri-infarct region
is great than about 4 mg.
57. The method of claim 55, wherein the volume of solution of SDF-1
plasmid administered to the weakened, ischemic, and/or peri-infarct
region is at least about 10 ml.
58. The method of claim 51, wherein the SDF-1 plasmid is
administered to the weakened, ischemic, and/or peri-infarct region
in at least 10 injections of the solution with each injection
comprising about 0.33 mg/ml to about 5 mg/ml of SDF-1
plasmid/solution and each injection has a volume of at least about
0.2 ml.
59. The method of claim 51, wherein SDF-1 is expressed at a
therapeutically effective amount in the weakened, ischemic, and/or
peri-infarct region for greater than about three days.
60. The method of claim 41, wherein the myocardial tissue of the
subject is imaged to define the area of weakened, ischemic, and/or
peri-infarct region prior to administration of the SDF-1 plasmid
and the SDF-1 plasmid is administered to the weakened, ischemic,
and/or peri-infarct region defined by the imaging.
61. The method of claim 60, wherein the imaging includes at least
one of echocardiography, magnetic resonance imaging, coronary
angiogram, electroanatomical mapping, or fluoroscopy.
62. A method of improving left ventricular end systolic volume in a
large mammal after myocardial infarction comprising: injecting a
solution comprising SDF-1 expressing plasmid DNA into the
peri-infarct region of the mammal by catheterization, the SDF-1
being expressed from the peri-infarct region at an amount effective
to cause functional improvement in left ventricular end systolic
volume.
63. The method of claim 62, wherein the amount of SDF-1 injected
into the peri-infarct region is effective to improve left
ventricular end systolic volume of at least about 10%.
64. The method of claim 62, wherein the amount of SDF-1 injected
into the peri-infarct region is effective to improve left
ventricular ejection fraction by at least about 10%.
65. The method of claim 62, wherein the amount of SDF-1 injected
into the peri-infarct region is effective to improve 6 minute walk
distance by at least about 30 meters or improve NYHA class by at
least 1 class.
66. The method of claim 62, wherein the amount of SDF-1 injected
into the peri-infarct region is effective to improve left
ventricular ejection fraction by at least about 10%, improve wall
motion score index by about 5%, improve 6 minute walk distance by
at least about 30 meters, and improve NYHA class by at least 1
class.
67. The method of claim 62, wherein the SDF-1 plasmid solution is
injected into the peri-infarct region in multiple injections of the
solution with each injection comprising about 0.33 mg/ml to about 5
mg/ml of SDF-1 plasmid/solution.
68. The method of claim 67, wherein each injection has a volume of
at least about 0.2 ml.
69. The method of claim 68, wherein the SDF-1 plasmid is
administered to the peri-infarct region in at least about 10
injections.
70. The method of claim 69, wherein the amount of SDF-1 plasmid
administered to the peri-infarct region is great than about 4
mg.
71. The method of claim 70 wherein the volume of solution of SDF-1
plasmid administered to the peri-infarct region is at least about
10 ml.
72. The method of claim 62, wherein the SDF-1 plasmid is injected
into the peri-infarct region in at least 10 injections of the
solution with each injection comprising about 0.33 mg/ml to about 5
mg/ml of SDF-1 plasmid/solution and each injection has a volume of
at least about 0.2 ml.
73. The method of claim 62, wherein the myocardial tissue of the
subject is imaged to define the peri-infarct region prior to
injection of the SDF-1 plasmid and the SDF-1 plasmid is injected
into the peri-infarct region defined by the imaging.
74. The method of claim 73, wherein the imaging includes at least
one of echocardiography, magnetic resonance imaging, coronary
angiogram, electroanatomical mapping, or fluoroscopy.
Description
RELATED APPLICATION
[0001] This application claims priority from U.S. Provisional
Application Nos. 61/237,775, filed Aug. 28, 2009, and 61/334,216,
filed May 13, 2010, the subject matter which is incorporated herein
by reference in its entirety.
FIELD OF THE INVENTION
[0002] This application relates to SDF-1 delivery methods and
compositions for treating a cardiomyopathy and to the use of SDF-1
delivery methods and compositions for treating an ischemic
cardiomyopathy.
BACKGROUND OF THE INVENTION
[0003] Ischemia is a condition wherein the blood flow is completely
obstructed or considerably reduced in localized parts of the body,
resulting in anoxia, reduced supply of substrates and accumulation
of metabolites. Although the extent of ischemia depends on the
acuteness of vascular obstruction, its duration, tissue sensitivity
to it, and developmental extent of collateral vessels, dysfunction
usually occurs in ischemic organs or tissues, and prolonged
ischemia results in atrophy, denaturation, apoptosis, and necrosis
of affected tissues.
[0004] In ischemic cardiomyopathy, which are diseases that affect
the coronary artery and cause myocardial ischemia, the extent of
ischemic myocardial cell injury proceeds from reversible cell
damage to irreversible cell damage with increasing time of the
coronary artery obstruction.
SUMMARY OF THE INVENTION
[0005] This application relates to a method of treating a
cardiomyopathy in a subject. The cardiomyopathy can include, for
example, cardiomyopathies associated with a pulmonary embolus, a
venous thrombosis, a myocardial infarction, a transient ischemic
attack, a peripheral vascular disorder, atherosclerosis, and/or
other myocardial injury or vascular disease. The method includes
administering directly to or expressing locally in a weakened,
ischemic, and/or peri-infarct region of myocardial tissue of the
subject an amount of SDF-1 effective to cause functional
improvement in at least one of the following parameters: left
ventricular volume, left ventricular area, left ventricular
dimension, cardiac function, 6-minute walk test (6MWT), or New York
Heart Association (NYHA) functional classification.
[0006] In an aspect of the application, the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to cause functional improvement in at least one of
left ventricular end systolic volume, left ventricular ejection
fraction, wall motion score index, left ventricular end diastolic
length, left ventricular end systolic length, left ventricular end
diastolic area, left ventricular end systolic area, left
ventricular end diastolic volume, 6-minute walk test (6MWT), or New
York Heart Association (NYHA) functional classification. In another
aspect of the application, the amount of SDF-1 administered to the
weakened, ischemic, and/or peri-infarct region is effective to
improve left ventricular end systolic volume. In a further aspect
of the application, the amount of SDF-1 administered to the
weakened, ischemic, and/or peri-infarct region is effective to
improve left ventricular ejection fraction.
[0007] In some aspects of the application, the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to improve left ventricular end systolic volume by at
least about 10%. In other aspects of the application, the amount of
SDF-1 administered to the weakened, ischemic, and/or peri-infarct
region is effective to improve left ventricular end systolic volume
by at least about 15%. In still further aspects of the application,
the amount of SDF-1 administered to the weakened, ischemic, and/or
peri-infarct region is effective to improve left ventricular end
systolic volume by at least about 10%, improve left ventricular
ejection fraction by at least about 10%, improve wall motion score
index by at least about 5%, improve six minute walk distance at
least about 30 meters, and improve NYHA class by at least 1 class.
In a further aspect of the application, the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to improve left ventricular ejection fraction by at
least about 10%.
[0008] In another aspect of the application, the amount of SDF-1
administered to the weakened, ischemic, and/or peri-infarct region
is effective to substantially improve vasculogenesis of the
weakened, ischemic, and/or peri-infarct region by at least about
20% based on vessel density or measured by myocardial perfusion
imaging (e.g., SPECT or PET) with an improvement in summed rest
score, summed stress score, and/or summed difference score of at
least about 10%. The SDF-1 can be administered by injecting a
solution comprising SDF-1 expressing plasmid in the weakened,
ischemic, and/or peri-infarct region and expressing SDF-1 from the
weakened, ischemic, and/or peri-infarct region. The SDF-1 can be
expressed from the weakened, ischemic, and/or peri-infarct region
at an amount effective to improve left ventricular end systolic
volume.
[0009] In an aspect of the application, the SDF-1 plasmid can be
administered to the weakened, ischemic, and/or peri-infarct region
in multiple injections of the solution with each injection
comprising about 0.33 mg/ml to about 5 mg/ml of SDF-1 plasmid
solution. In one example, the SDF-1 plasmid can be administered to
the weakened, ischemic, and/or peri-infarct region in at least
about 10 injections. Each injection administered to the weakened,
ischemic, and/or peri-infarct region can have a volume of at least
about 0.2 ml. The SDF-1 can be expressed in the weakened, ischemic,
and/or peri-infarct region for greater than about three days.
[0010] In an example application, each injection of solution
comprising SDF-1 expressing plasmid can have an injection volume of
at least about 0.2 ml and an SDF-1 plasmid concentration per
injection of about 0.33 mg/ml to about 5 mg/ml. In another aspect
of the application, at least one functional parameter of the of the
heart can be improved by injecting the SDF-1 plasmid into the
weakened, ischemic, and/or peri-infarct region of the heart at an
injection volume per site of at least about 0.2 ml, in at least
about 10 injection sites, and at an SDF-1 plasmid concentration per
injection of about 0.33 mg/ml to about 5 mg/ml.
[0011] In a further example, the amount of SDF-1 plasmid
administered to the weakened, ischemic, and/or peri-infarct region
that can improve at least one functional parameter of the heart is
greater than about 4 mg. The volume of solution of SDF-1 plasmid
administered to the weakened, ischemic, and/or peri-infarct region
that can improve at least one functional parameter of the heart is
at least about 10 ml.
[0012] In another aspect of the application, the subject to which
the SDF-1 is administered can be a large mammal, such as a human or
pig. The SDF-1 plasmid can be administered to the subject by
catheterization, such as intra-coronary catheterization or
endo-ventricular catheterization. The myocardial tissue of the
subject can be imaged to define the area of weakened, ischemic,
and/or peri-infarct region prior to administration of the SDF-1
plasmid, and the SDF-1 plasmid can be administered to the weakened,
ischemic, and/or peri-infarct region defined by the imaging. The
imaging can include at least one of echocardiography, magnetic
resonance imaging, coronary angiogram, electroanatomical mapping,
or fluoroscopy.
[0013] The application also relates to a method of treating a
myocardial infarction in a large mammal by administering SDF-1
plasmid to the peri-infarct region of the myocardium of the mammal
by catheterization, such as intra-coronary catheterization or
endo-ventricular catheterization. The SDF-1 administered by
catheterization can be expressed from the peri-infarct region at an
amount effective to cause functional improvement in at least one of
the following parameters: left ventricular volume, left ventricular
area, left ventricular dimension, cardiac function, 6-minute walk
test (6MWT), or New York Heart Association (NYHA) functional
classification.
[0014] In an aspect of the application, the amount of SDF-1
administered to the peri-infarct region is effective to cause
functional improvement in at least one of left ventricular end
systolic volume, left ventricular ejection fraction, wall motion
score index, left ventricular end diastolic length, left
ventricular end systolic length, left ventricular end diastolic
area, left ventricular end systolic area, left ventricular end
diastolic volume, 6-minute walk test (6MWT), or New York Heart
Association (NYHA) functional classification. In another aspect of
the application, the amount of SDF-1 administered to the
peri-infarct region is effective to improve left ventricular end
systolic volume. In a further aspect of the application, the amount
of SDF-1 administered to the weakened, ischemic, and/or
peri-infarct region is effective to improve left ventricular
ejection fraction.
[0015] In some aspects of the application, the amount of SDF-1
administered to the peri-infarct region is effective to improve
left ventricular end systolic volume by at least about 10%. In
other aspects of the application, the amount of SDF-1 administered
to the peri-infarct region is effective to improve left ventricular
end systolic volume by at least about 15%. In still further aspects
of the application, the amount of SDF-1 administered to the
peri-infarct region is effective to improve left ventricular end
systolic volume by at least about 10%, improve left ventricular
ejection fraction by at least about 10%, improve wall motion score
index by about 5%, improve six minute walk distance at least about
30 meters, or improve NYHA class by at least 1 class. In a further
aspect of the application, the amount of SDF-1 administered to the
weakened, ischemic, and/or peri-infarct region is effective to
improve left ventricular ejection fraction by at least about
10%.
[0016] In another aspect of the application, the amount of SDF-1
administered to the peri-infarct region is effective to
substantially improve vasculogenesis of the peri-infarct region by
at least about 20% based on vessel density.
[0017] In an aspect of the application, the SDF-1 plasmid can be
administered to the weakened, ischemic, and/or peri-infarct region
in multiple injections of the solution with each injection
comprising about 0.33 mg/ml to about 5 mg/ml of SDF-1
plasmid/solution. In one example, the SDF-1 plasmid can be
administered to the weakened, ischemic, and/or peri-infarct region
in at least about 10 injections. Each injection administered to the
weakened, ischemic, and/or peri-infarct region can have a volume of
at least about 0.2 ml. The SDF-1 can be expressed in the weakened,
ischemic, and/or peri-infarct region for greater than about three
days.
[0018] In an example application, each injection of solution
comprising SDF-1 expressing plasmid can have an injection volume of
at least about 0.2 ml and an SDF-1 plasmid concentration per
injection of about 0.33 mg/ml to about 5 mg/ml. In another aspect
of the application, at least one functional parameter of the of the
heart can be improved by injecting the SDF-1 plasmid into the
weakened, ischemic, and/or peri-infarct region of the heart at an
injection volume per site of at least about 0.2 ml, in at least
about 10 injection sites, and at an SDF-1 plasmid concentration per
injection of about 0.33 mg/ml to about 5 mg/ml.
[0019] In a further example, the amount of SDF-1 plasmid
administered to the weakened, ischemic, and/or peri-infarct region
that can improve at least one functional parameter of the heart is
greater than about 4 mg. The volume of solution of SDF-1 plasmid
administered to the weakened, ischemic, and/or peri-infarct region
that can improve at least one functional parameter of the heart is
at least about 10 ml.
[0020] The application further relates to a method of improving
left ventricular end systolic volume in a large mammal after
myocardial infarction. The method includes administering SDF-1
plasmid to the peri-infarct region of the mammal by
endo-ventricular catheterization. The SDF-1 can be expressed from
the pen-infarct region at an amount effective to cause functional
improvement in left ventricular end systolic volume.
[0021] In some aspects of the application, the amount of SDF-1
administered to the peri-infarct region is effective to improve
left ventricular end systolic volume by at least about 10%. In
other aspects of the application, the amount of SDF-1 administered
to the peri-infarct region is effective to improve left ventricular
end systolic volume by at least about 15%. In still further aspects
of the application, the amount of SDF-1 administered to the
peri-infarct region is effective to improve left ventricular end
systolic volume by at least about 10%, improve left ventricular
ejection fraction by at least about 10%, improve wall motion score
index by about 5%, improve six minute walk distance at least about
30 meters, or improve NYHA class by at least 1 class.
[0022] In an aspect of the application, the SDF-1 plasmid can be
administered to the weakened, ischemic, and/or peri-infarct region
in multiple injections of the solution with each injection
comprising about 0.33 mg/ml to about 5 mg/ml of SDF-1
plasmid/solution. In one example, the SDF-1 plasmid can be
administered to the weakened, ischemic, and/or peri-infarct region
in at least about 10 injections. Each injection administered to the
weakened, ischemic, and/or peri-infarct region can have a volume of
at least about 0.2 ml. The SDF-1 can be expressed in the weakened,
ischemic, and/or peri-infarct region for greater than about three
days.
[0023] In an example application, each injection of solution
comprising SDF-1 expressing plasmid can have an injection volume of
at least about 0.2 ml and an SDF-1 plasmid concentration per
injection of about 0.33 mg/ml to about 5 mg/ml. In another aspect
of the application, left ventricular end systolic volume of the of
the heart can be improved can be improved at about 10% by injecting
the SDF-1 plasmid into the weakened, ischemic, and/or peri-infarct
region of the heart at an injection volume per site of at least
about 0.2 ml, in at least about 10 injection sites, and at an SDF-1
plasmid concentration per injection of about 0.33 mg/ml to about 5
mg/ml.
[0024] In a further example, the amount of SDF-1 plasmid
administered to the weakened, ischemic, and/or peri-infarct region
that can improve left ventricular end systolic volume is greater
than about 4 mg. The volume of solution of SDF-1 plasmid
administered to the weakened, ischemic, and/or peri-infarct region
that can improve left ventricular end systolic volume of the heart
is at least about 10 ml.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The foregoing and other features of the application will
become apparent to those skilled in the art to which the
application relates upon reading the following description with
reference to the accompanying drawings.
[0026] FIG. 1 is a chart illustrating luciferase expression for
varying amounts and volume of DNA in a porcine model;
[0027] FIG. 2 is a chart illustrating % change of left ventricular
end systolic volume for various amounts of SDF-1 plasmid using a
porcine model of congestive heart failure 30 days following SDF-1
injection;
[0028] FIG. 3 is a chart illustrating % change of left ventricular
ejection fraction for various amounts of SDF-1 plasmid using a
porcine model of congestive heart failure 30 days following SDF-1
injection;
[0029] FIG. 4 is a chart illustrating % change in wall motion score
index for various amounts of SDF-1 plasmid using a porcine model of
congestive heart failure 30 days following SDF-1 injection;
[0030] FIG. 5 is a chart illustrating % change of left ventricular
end systolic volume for various amounts of SDF-1 plasmid using a
porcine model of congestive heart failure 90 days following SDF-1
injection; and
[0031] FIG. 6 is a chart illustrating % change of vessel density
for various amounts of SDF-1 plasmid using a porcine model of
congestive heart failure 30 days following SDF-1 injection.
[0032] FIG. 7 is a schematic diagram of a plasmid vector in
accordance with an aspect of the application.
[0033] FIG. 8 is an image showing plasmid expression over a
substantial portion of a porcine heart.
[0034] FIG. 9 is a chart illustrating left ventricular end systolic
volume at baseline and 30 days post-initial injection. All groups
show similar increases in left ventricular end systolic volume at
30 days. N=3 for all data points. Data presented as
mean.+-.SEM.
[0035] FIG. 10 is a chart illustrating left ventricular ejection
fraction at baseline and 30 days post-initial injection. All groups
show lack of improvement in left ventricular ejection fraction. N=3
for all data points. Data presented as mean.+-.SEM.
DETAILED DESCRIPTION
[0036] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the application(s) belong. All
patents, patent applications, published applications and
publications, Genbank sequences, websites and other published
materials referred to throughout the entire disclosure herein,
unless noted otherwise, are incorporated by reference in their
entirety. In the event that there are a plurality of definitions
for terms herein, those in this section prevail. Unless otherwise
defined, all technical terms used herein have the same meaning as
commonly understood by one of ordinary skill in the art to which
this application belongs. Commonly understood definitions of
molecular biology terms can be found in, for example, Rieger et
al., Glossary of Genetics: Classical and Molecular, 5th edition,
Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford
University Press: New York, 1994.
[0037] Methods involving conventional molecular biology techniques
are described herein. Such techniques are generally known in the
art and are described in detail in methodology treatises, such as
Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed.
Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed.
Ausubel et al., Greene Publishing and Wiley-Interscience, New York,
1992 (with periodic updates). Methods for chemical synthesis of
nucleic acids are discussed, for example, in Beaucage and
Carruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucci et al.,
J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleic
acids can be performed, for example, on commercial automated
oligonucleotide synthesizers. Immunological methods (e.g.,
preparation of antigen-specific antibodies, immunoprecipitation,
and immunoblotting) are described, e.g., in Current Protocols in
Immunology, ed. Coligan et al., John Wiley & Sons, New York,
1991; and Methods of Immunological Analysis, ed. Masseyeff et al.,
John Wiley & Sons, New York, 1992. Conventional methods of gene
transfer and gene therapy can also be adapted for use in the
application. See, e.g., Gene Therapy: Principles and Applications,
ed. T. Blackenstein, Springer Verlag, 1999; Gene Therapy Protocols
(Methods in Molecular Medicine), ed. P. D. Robbins, Humana Press,
1997; and Retro-vectors for Human Gene Therapy, ed. C. P. Hodgson,
Springer Verlag, 1996.
[0038] Where reference is made to a URL or other such identifier or
address, it understood that such identifiers can change and
particular information on the internet can come and go, but
equivalent information can be found by searching the internet.
Reference thereto evidences the availability and public
dissemination of such information.
[0039] As used herein, "nucleic acid" refers to a polynucleotide
containing at least two covalently linked nucleotide or nucleotide
analog subunits. A nucleic acid can be a deoxyribonucleic acid
(DNA), a ribonucleic acid (RNA), or an analog of DNA or RNA.
Nucleotide analogs are commercially available and methods of
preparing polynucleotides containing such nucleotide analogs are
known (Lin et al. (1994) Nucl. Acids Res. 22:5220-5234; Jellinek et
al. (1995) Biochemistry 34:11363-11372; Pagratis et al. (1997)
Nature Biotechnol. 15:68-73). The nucleic acid can be
single-stranded, double-stranded, or a mixture thereof. For
purposes herein, unless specified otherwise, the nucleic acid is
double-stranded, or it is apparent from the context.
[0040] As used herein, "DNA" is meant to include all types and
sizes of DNA molecules including cDNA, plasmids and DNA including
modified nucleotides and nucleotide analogs.
[0041] As used herein, "nucleotides" include nucleoside mono-, di-,
and triphosphates. Nucleotides also include modified nucleotides,
such as, but are not limited to, phosphorothioate nucleotides and
deazapurine nucleotides and other nucleotide analogs.
[0042] As used herein, the term "subject" or "patient" refers to
animals into which the large DNA molecules can be introduced.
Included are higher organisms, such as mammals and birds, including
humans, primates, rodents, cattle, pigs, rabbits, goats, sheep,
mice, rats, guinea pigs, cats, dogs, horses, chicken and
others.
[0043] As used herein "large mammal" refers to mammals having a
typical adult weight of at least 10 kg. Such large mammals can
include, for example, humans, primates, dogs, pigs, cattle and is
meant to exclude smaller mammals, such as mice, rats, guinea pigs,
and other rodents.
[0044] As used herein, "administering to a subject" is a procedure
by which one or more delivery agents and/or large nucleic acid
molecules, together or separately, are introduced into or applied
onto a subject such that target cells which are present in the
subject are eventually contacted with the agent and/or the large
nucleic acid molecules.
[0045] As used herein, "delivery," which is used interchangeably
with "transduction," refers to the process by which exogenous
nucleic acid molecules are transferred into a cell such that they
are located inside the cell. Delivery of nucleic acids is a
distinct process from expression of nucleic acids.
[0046] As used herein, a "multiple cloning site (MCS)" is a nucleic
acid region in a plasmid that contains multiple restriction enzyme
sites, any of which can be used in conjunction with standard
recombinant technology to digest the vector. "Restriction enzyme
digestion" refers to catalytic cleavage of a nucleic acid molecule
with an enzyme that functions only at specific locations in a
nucleic acid molecule. Many of these restriction enzymes are
commercially available. Use of such enzymes is widely understood by
those of skill in the art. Frequently, a vector is linearized or
fragmented using a restriction enzyme that cuts within the MCS to
enable exogenous sequences to be ligated to the vector.
[0047] As used herein, "origin of replication" (often termed
"ori"), is a specific nucleic acid sequence at which replication is
initiated. Alternatively, an autonomously replicating sequence
(ARS) can be employed if the host cell is yeast.
[0048] As used herein, "selectable or screenable markers" confer an
identifiable change to a cell permitting easy identification of
cells containing an expression vector. Generally, a selectable
marker is one that confers a property that allows for selection. A
positive selectable marker is one in which the presence of the
marker allows for its selection, while a negative selectable marker
is one in which its presence prevents its selection. An example of
a positive selectable marker is a drug resistance marker.
[0049] Usually the inclusion of a drug selection marker aids in the
cloning and identification of transformants, for example, genes
that confer resistance to neomycin, puromycin, hygromycin, DHFR,
GPT, zeocin and histidinol are useful selectable markers. In
addition to markers conferring a phenotype that allows for the
discrimination of transformants based on the implementation of
conditions, other types of markers including screenable markers
such as GFP, whose basis is calorimetric analysis, are also
contemplated. Alternatively, screenable enzymes such as herpes
simplex virus thymidine kinase (tk) or chloramphenicol
acetyltransferase (CAT) may be utilized. One of skill in the art
would also know how to employ immunologic markers, possibly in
conjunction with FACS analysis. The marker used is not believed to
be important, so long as it is capable of being expressed
simultaneously with the nucleic acid encoding a gene product.
Further examples of selectable and screenable markers are well
known to one of skill in the art.
[0050] The term "transfection" is used to refer to the uptake of
foreign DNA by a cell. A cell has been "transfected" when exogenous
DNA has been introduced inside the cell membrane. A number of
transfection techniques are generally known in the art. See, e.g.,
Graham et al., Virology 52:456 (1973); Sambrook et al., Molecular
Cloning: A Laboratory Manual (1989); Davis et al., Basic Methods in
Molecular Biology (1986); Chu et al., Gene 13:197 (1981). Such
techniques can be used to introduce one or more exogenous DNA
moieties, such as a nucleotide integration vector and other nucleic
acid molecules, into suitable host cells. The term captures
chemical, electrical, and viral-mediated transfection
procedures.
[0051] As used herein, "expression" refers to the process by which
nucleic acid is translated into peptides or is transcribed into
RNA, which, for example, can be translated into peptides,
polypeptides or proteins. If the nucleic acid is derived from
genomic DNA, expression may, if an appropriate eukaryotic host cell
or organism is selected, include splicing of the mRNA. For
heterologous nucleic acid to be expressed in a host cell, it must
initially be delivered into the cell and then, once in the cell,
ultimately reside in the nucleus.
[0052] As used herein, "genetic therapy" involves the transfer of
heterologous DNA to cells of a mammal, particularly a human, with a
disorder or conditions for which therapy or diagnosis is sought.
The DNA is introduced into the selected target cells in a manner
such that the heterologous DNA is expressed and a therapeutic
product encoded thereby is produced. Alternatively, the
heterologous DNA may in some manner mediate expression of DNA that
encodes the therapeutic product; it may encode a product, such as a
peptide or RNA that in some manner mediates, directly or
indirectly, expression of a therapeutic product. Genetic therapy
may also be used to deliver nucleic acid encoding a gene product to
replace a defective gene or supplement a gene product produced by
the mammal or the cell in which it is introduced. The introduced
nucleic acid may encode a therapeutic compound, such as a growth
factor inhibitor thereof, or a tumor necrosis factor or inhibitor
thereof, such as a receptor therefore, that is not normally
produced in the mammalian host or that is not produced in
therapeutically effective amounts or at a therapeutically useful
time. The heterologous DNA encoding the therapeutic product may be
modified prior to introduction into the cells of the afflicted host
in order to enhance or otherwise alter the product or expression
thereof.
[0053] As used herein, "heterologous nucleic acid sequence" is
typically DNA that encodes RNA and proteins that are not normally
produced in vivo by the cell in which it is expressed or that
mediates or encodes mediators that alter expression of endogenous
DNA by affecting transcription, translation, or other regulatable
biochemical processes. A heterologous nucleic acid sequence may
also be referred to as foreign DNA. Any DNA that one of skill in
the art would recognize or consider as heterologous or foreign to
the cell in which it is expressed is herein encompassed by
heterologous DNA. Examples of heterologous DNA include, but are not
limited to, DNA that encodes traceable marker proteins, such as a
protein that confers drug resistance, DNA that encodes
therapeutically effective substances, such as anti-cancer agents,
enzymes and hormones, and DNA that encodes other types of proteins,
such as antibodies. Antibodies that are encoded by heterologous DNA
may be secreted or expressed on the surface of the cell in which
the heterologous DNA has been introduced.
[0054] As used herein the term "cardiomyopathy" refers to the
deterioration of the function of the myocardium (i.e., the actual
heart muscle) for any reason. Subjects with cardiomyopathy are
often at risk of arrhythmia, sudden cardiac death, or
hospitalization or death due to heart failure.
[0055] As used herein, the term "ischemic cardiomyopathy" is a
weakness in the muscle of the heart due to inadequate oxygen
delivery to the myocardium with coronary artery disease being the
most common cause.
[0056] As used herein the term "ischemic cardiac disease" refers to
any condition in which heart muscle is damaged or works
inefficiently because of an absence or relative deficiency of its
blood supply; most often caused by atherosclerosis, it includes
angina pectoris, acute myocardial infarction, chronic ischemic
heart disease, and sudden death.
[0057] As used herein the term "myocardial infarction" refers to
the damaging or death of an area of the heart muscle (myocardium)
resulting from a blocked blood supply to that area.
[0058] As used herein the term "6-minute walk test" or "6MWT"
refers to a test that measures the distance that a patient can
quickly walk on a flat, hard surface in a period of 6 minutes (the
6MWD). It evaluates the global and integrated responses of all the
systems involved during exercise, including the pulmonary and
cardiovascular systems, systemic circulation, peripheral
circulation, blood, neuromuscular units, and muscle metabolism. It
does not provide specific information on the function of each of
the different organs and systems involved in exercise or the
mechanism of exercise limitation, as is possible with maximal
cardiopulmonary exercise testing. The self-paced 6MWT assesses the
submaximal level of functional capacity. (See for example, AM J
Respir Crit Care Med, Vol. 166. Pp 111-117 (2002))
[0059] As used herein "New York Heart Association (NYHA) functional
classification" refers to a classification for the extent of heart
failure. It places patients in one of four categories based on how
much they are limited during physical activity; the
limitations/symptoms are in regards to normal breathing and varying
degrees in shortness of breath and or angina pain:
TABLE-US-00001 NYHA Class Symptoms I No symptoms and no limitation
in ordinary physical activity, e.g. shortness of breath when
walking, climbing stairs etc. II Mild symptoms (mild shortness of
breath and/or angina) and slight limitation during ordinary
activity. III Marked limitation in activity due to symptoms, even
during less-than-ordinary activity, e.g. walking short distances
(20-100 m). Comfortable only at rest. IV Severe limitations.
Experiences symptoms even while at rest. Mostly bedbound
patients.
[0060] This application relates to compositions and methods of
treating a cardiomyopathy in a subject that results in reduced
and/or impaired myocardial function. The cardiomyopathy treated by
the compositions and methods herein can include cardiomyopathies
associated with a pulmonary embolus, a venous thrombosis, a
myocardial infarction, a transient ischemic attack, a peripheral
vascular disorder, atherosclerosis, ischemic cardiac disease and/or
other myocardial injury or vascular disease. The method of treating
the cardiomyopathy can include locally administering (or locally
delivering) to weakened myocardial tissue, ischemic myocardial
tissue, and/or apoptotic myocardial tissue, such as the
peri-infarct region of a heart following myocardial infarction, an
amount of stromal-cell derived factor-1 (SDF-1) that is effective
to cause functional improvement in at least one of the following
parameters: left ventricular volume, left ventricular area, left
ventricular dimension, cardiac function, 6-minute walk test (6MWT),
or New York Heart Association (NYHA) functional classification.
[0061] It was found using a porcine model of heart failure that
mimics heart failure in a human that functional improvement of
ischemic myocardial tissue is dependent on the amount, dose, and/or
delivery of SDF-1 administered to the ischemic myocardial tissue
and that the amount, dose, and/or delivery of SDF-1 to the ischemic
myocardial tissue can be optimized so that myocardial functional
parameters, such as left ventricular volume, left ventricular area,
left ventricular dimension, or cardiac function are substantially
improved. As discussed below, in some aspects, the amount,
concentration, and volume of SDF-1 administered to the ischemic
myocardial tissue can be controlled and/or optimized to
substantially improve the functional parameters (e.g., left
ventricular volume, left ventricular area, left ventricular
dimension, cardiac function, 6-minute walk test (6MWT), and/or New
York Heart Association (NYHA) functional classification) while
mitigating adverse side effects.
[0062] In one example, the SDF-1 can be administered directly or
locally to a weakened region, an ischemic region, and/or
peri-infarct region of myocardial tissue of a large mammal (e.g.,
pig or human) in which there is a deterioration or worsening of a
functional parameter of the heart, such as left ventricular volume,
left ventricular area, left ventricular dimension, or cardiac
function as a result of an ischemic cardiomyopathy, such as a
myocardial infarction. The deterioration or worsening of the
functional parameter can include, for example, an increase in left
ventricular end systolic volume, decrease in left ventricular
ejection fraction, increase in wall motion score index, increase in
left ventricular end diastolic length, increase in left ventricular
end systolic length, increase in left ventricular end diastolic
area (e.g., mitral valve level and papillary muscle insertion
level), increase in left ventricular end systolic area (e.g.,
mitral valve level and papillary muscle insertion level), or
increase in left ventricular end diastolic volume as measured
using, for example, using echocardiography.
[0063] In an aspect of the application, the amount of SDF-1
administered to the weakened region, ischemic region, and/or
peri-infarct region of the myocardial tissue of the large mammal
can be an amount effective to improve at least one functional
parameter of the myocardium, such as a decrease in left ventricular
end systolic volume, increase in left ventricular ejection
fraction, decrease in wall motion score index, decrease in left
ventricular end diastolic length, decrease in left ventricular end
systolic length, decrease in left ventricular end diastolic area
(e.g., mitral valve level and papillary muscle insertion level),
decrease in left ventricular end systolic area (e.g., mitral valve
level and papillary muscle insertion level), or decrease in left
ventricular end diastolic volume measured using, for example, using
echocardiography as well as improve the subject's 6-minute walk
test (6MWT) or New York Heart Association (NYHA) functional
classification.
[0064] In another aspect of the application, the amount of SDF-1
administered to the weakened region, ischemic region, and/or
peri-infarct region of the myocardial tissue of the large mammal
with a cardiomyopathy is effective to improve left ventricular end
systolic volume in the mammal by at least about 10%, and more
specifically at least about 15%, after 30 days following
administration as measured by echocardiography. The percent
improvement is relative to each subject treated and is based on the
respective parameter measured prior to or at the time of
therapeutic intervention or treatment.
[0065] In a further aspect of the application, the amount of SDF-1
administered to the weakened region, ischemic region, and/or
peri-infarct region of the myocardial tissue of the large mammal
with a cardiomyopathy is effective to improve left ventricular end
systolic volume by at least about 10%, improve left ventricular
ejection fraction by at least about 10%, and improve wall motion
score index by about 5%, after 30 days following administration as
measured by echocardiography.
[0066] In a still further aspect of the application, the amount of
SDF-1 administered to the weakened region, ischemic region, and/or
peri-infarct region of the myocardial tissue of the large mammal
with a cardiomyopathy is effective to improve vasculogenesis of the
weakened region, ischemic region, and/or peri-infarct region by at
least 20% based on vessel density or an increase in cardiac
perfusion measured by SPECT imaging. A 20% improvement in
vasculogenesis has been shown to be clinically significant (Losordo
Circulation 2002; 105:2012).
[0067] In a still further aspect of the application, the amount of
SDF-1 administered to the weakened region, ischemic region, and/or
peri-infarct region of the myocardial tissue of the large mammal
with a cardiomyopathy is effective to improve six minute walk
distance at least about 30 meters or improve NYHA class by at least
1 class.
[0068] The SDF-1 described herein can be administered to the
weakened region, the ischemic region, and/or peri-infarct region of
the myocardial tissue following tissue injury (e.g., myocardial
infarction) to about hours, days, weeks, or months after onset of
down-regulation of SDF-1. The period of time that the SDF-1 is
administered to the cells can comprise from about immediately after
onset of the cardiomyopathy (e.g., myocardial infarction) to about
days, weeks, or months after the onset of the ischemic disorder or
tissue injury.
[0069] SDF-1 in accordance with the application that is
administered to the weakened, ischemic, and/or a peri-infarct
region of the myocardial tissue peri-infarct region can have an
amino acid sequence that is substantially similar to a native
mammalian SDF-1 amino acid sequence. The amino acid sequence of a
number of different mammalian SDF-1 protein are known including
human, mouse, and rat. The human and rat SDF-1 amino acid sequences
are at least about 92% identical (e.g., about 97% identical). SDF-1
can comprise two isoforms, SDF-1 alpha and SDF-1 beta, both of
which are referred to herein as SDF-1 unless identified
otherwise.
[0070] The SDF-1 can have an amino acid sequence substantially
identical to SEQ ID NO: 1. The SDF-1 that is over-expressed can
also have an amino acid sequence substantially similar to one of
the foregoing mammalian SDF-1 proteins. For example, the SDF-1 that
is over-expressed can have an amino acid sequence substantially
similar to SEQ ID NO: 2. SEQ ID NO: 2, which substantially
comprises SEQ ID NO: 1, is the amino acid sequence for human SDF-1
and is identified by GenBank Accession No. NP954637. The SDF-1 that
is over-expressed can also have an amino acid sequence that is
substantially identical to SEQ ID NO: 3. SEQ ID NO: 3 includes the
amino acid sequences for rat SDF and is identified by GenBank
Accession No. AAF01066.
[0071] The SDF-1 in accordance with the application can also be a
variant of mammalian SDF-1, such as a fragment, analog and
derivative of mammalian SDF-1. Such variants include, for example,
a polypeptide encoded by a naturally occurring allelic variant of
native SDF-1 gene (i.e., a naturally occurring nucleic acid that
encodes a naturally occurring mammalian SDF-1 polypeptide), a
polypeptide encoded by an alternative splice form of a native SDF-1
gene, a polypeptide encoded by a homolog or ortholog of a native
SDF-1 gene, and a polypeptide encoded by a non-naturally occurring
variant of a native SDF-1 gene.
[0072] SDF-1 variants have a peptide sequence that differs from a
native SDF-1 polypeptide in one or more amino acids. The peptide
sequence of such variants can feature a deletion, addition, or
substitution of one or more amino acids of a SDF-1 variant Amino
acid insertions are preferably of about 1 to 4 contiguous amino
acids, and deletions are preferably of about 1 to 10 contiguous
amino acids. Variant SDF-1 polypeptides substantially maintain a
native SDF-1 functional activity. Examples of SDF-1 polypeptide
variants can be made by expressing nucleic acid molecules that
feature silent or conservative changes. One example of an SDF-1
variant is listed in U.S. Pat. No. 7,405,195, which is herein
incorporated by reference in its entirety.
[0073] SDF-1 polypeptide fragments corresponding to one or more
particular motifs and/or domains or to arbitrary sizes, are within
the scope of this application. Isolated peptidyl portions of SDF-1
can be obtained by screening peptides recombinantly produced from
the corresponding fragment of the nucleic acid encoding such
peptides. For example, an SDF-1 polypeptide may be arbitrarily
divided into fragments of desired length with no overlap of the
fragments, or preferably divided into overlapping fragments of a
desired length. The fragments can be produced recombinantly and
tested to identify those peptidyl fragments, which can function as
agonists of native CXCR-4 polypeptides.
[0074] Variants of SDF-1 polypeptides can also include recombinant
forms of the SDF-1 polypeptides. Recombinant polypeptides in some
embodiments, in addition to SDF-1 polypeptides, are encoded by a
nucleic acid that can have at least 70% sequence identity with the
nucleic acid sequence of a gene encoding a mammalian SDF-1.
[0075] SDF-1 variants can include agonistic forms of the protein
that constitutively express the functional activities of native
SDF-1. Other SDF-1 variants can include those that are resistant to
proteolytic cleavage, as for example, due to mutations, which alter
protease target sequences. Whether a change in the amino acid
sequence of a peptide results in a variant having one or more
functional activities of a native SDF-1 can be readily determined
by testing the variant for a native SDF-1 functional activity.
[0076] The SDF-1 nucleic acid that encodes the SDF-1 protein can be
a native or non-native nucleic acid and be in the form of RNA or in
the form of DNA (e.g., cDNA, genomic DNA, and synthetic DNA). The
DNA can be double-stranded or single-stranded, and if
single-stranded may be the coding (sense) strand or non-coding
(anti-sense) strand. The nucleic acid coding sequence that encodes
SDF-1 may be substantially similar to a nucleotide sequence of the
SDF-1 gene, such as nucleotide sequence shown in SEQ ID NO: 4 and
SEQ ID NO: 5. SEQ ID NO: 4 and SEQ ID NO: 5 comprise, respectively,
the nucleic acid sequences for human SDF-1 and rat SDF-1 and are
substantially similar to the nucleic sequences of GenBank Accession
No. NM199168 and GenBank Accession No. AF189724. The nucleic acid
coding sequence for SDF-1 can also be a different coding sequence
which, as a result of the redundancy or degeneracy of the genetic
code, encodes the same polypeptide as SEQ ID NO: 1, SEQ ID NO: 2,
and SEQ ID NO: 3.
[0077] Other nucleic acid molecules that encode SDF-1 are variants
of a native SDF-1, such as those that encode fragments, analogs and
derivatives of native SDF-1. Such variants may be, for example, a
naturally occurring allelic variant of a native SDF-1 gene, a
homolog or ortholog of a native SDF-1 gene, or a non-naturally
occurring variant of a native SDF-1 gene. These variants have a
nucleotide sequence that differs from a native SDF-1 gene in one or
more bases. For example, the nucleotide sequence of such variants
can feature a deletion, addition, or substitution of one or more
nucleotides of a native SDF-1 gene. Nucleic acid insertions are
preferably of about 1 to 10 contiguous nucleotides, and deletions
are preferably of about 1 to 10 contiguous nucleotides.
[0078] In other applications, variant SDF-1 displaying substantial
changes in structure can be generated by making nucleotide
substitutions that cause less than conservative changes in the
encoded polypeptide. Examples of such nucleotide substitutions are
those that cause changes in (a) the structure of the polypeptide
backbone; (b) the charge or hydrophobicity of the polypeptide; or
(c) the bulk of an amino acid side chain. Nucleotide substitutions
generally expected to produce the greatest changes in protein
properties are those that cause non-conservative changes in codons.
Examples of codon changes that are likely to cause major changes in
protein structure are those that cause substitution of (a) a
hydrophilic residue (e.g., serine or threonine), for (or by) a
hydrophobic residue (e.g., leucine, isoleucine, phenylalanine,
valine or alanine); (b) a cysteine or proline for (or by) any other
residue; (c) a residue having an electropositive side chain (e.g.,
lysine, arginine, or histidine), for (or by) an electronegative
residue (e.g., glutamine or aspartine); or (d) a residue having a
bulky side chain (e.g., phenylalanine), for (or by) one not having
a side chain, (e.g., glycine).
[0079] Naturally occurring allelic variants of a native SDF-1 gene
are nucleic acids isolated from mammalian tissue that have at least
70% sequence identity with a native SDF-1 gene, and encode
polypeptides having structural similarity to a native SDF-1
polypeptide. Homologs of a native SDF-1 gene are nucleic acids
isolated from other species that have at least 70% sequence
identity with the native gene, and encode polypeptides having
structural similarity to a native SDF-1 polypeptide. Public and/or
proprietary nucleic acid databases can be searched to identify
other nucleic acid molecules having a high percent (e.g., 70% or
more) sequence identity to a native SDF-1 gene.
[0080] Non-naturally occurring SDF-1 gene variants are nucleic
acids that do not occur in nature (e.g., are made by the hand of
man), have at least 70% sequence identity with a native SDF-1 gene,
and encode polypeptides having structural similarity to a native
SDF-1 polypeptide. Examples of non-naturally occurring SDF-1 gene
variants are those that encode a fragment of a native SDF-1
protein, those that hybridize to a native SDF-1 gene or a
complement of to a native SDF-1 gene under stringent conditions,
and those that share at least 65% sequence identity with a native
SDF-1 gene or a complement of a native SDF-1 gene.
[0081] Nucleic acids encoding fragments of a native SDF-1 gene in
some embodiments are those that encode amino acid residues of
native SDF-1. Shorter oligonucleotides that encode or hybridize
with nucleic acids that encode fragments of native SDF-1 can be
used as probes, primers, or antisense molecules. Longer
polynucleotides that encode or hybridize with nucleic acids that
encode fragments of a native SDF-1 can also be used in various
aspects of the application. Nucleic acids encoding fragments of a
native SDF-1 can be made by enzymatic digestion (e.g., using a
restriction enzyme) or chemical degradation of the full-length
native SDF-1 gene or variants thereof.
[0082] Nucleic acids that hybridize under stringent conditions to
one of the foregoing nucleic acids can also be used herein. For
example, such nucleic acids can be those that hybridize to one of
the foregoing nucleic acids under low stringency conditions,
moderate stringency conditions, or high stringency conditions.
[0083] Nucleic acid molecules encoding a SDF-1 fusion protein may
also be used in some embodiments. Such nucleic acids can be made by
preparing a construct (e.g., an expression vector) that expresses a
SDF-1 fusion protein when introduced into a suitable target cell.
For example, such a construct can be made by ligating a first
polynucleotide encoding a SDF-1 protein fused in frame with a
second polynucleotide encoding another protein such that expression
of the construct in a suitable expression system yields a fusion
protein.
[0084] The nucleic acids encoding SDF-1 can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, hybridization, etc. The nucleic
acids described herein may additionally include other appended
groups such as peptides (e.g., for targeting target cell receptors
in vivo), or agents facilitating transport across the cell
membrane, hybridization-triggered cleavage. To this end, the
nucleic acids may be conjugated to another molecule, (e.g., a
peptide), hybridization triggered cross-linking agent, transport
agent, hybridization-triggered cleavage agent, etc.
[0085] The SDF-1 can be delivered to the weakened, ischemic, and/or
peri-infarct region of the myocardial tissue by administering an
SDF-1 protein to the to the weakened, ischemic, and/or peri-infarct
region, or by introducing an agent into cells of the weakened
region, ischemic region, and/or peri-infarct region of the
myocardial tissue that causes, increases, and/or upregulates
expression of SDF-1 (i.e., SDF-1 agent). The SDF-1 protein
expressed from the cells can be an expression product of a
genetically modified cell.
[0086] The agent that causes, increases, and/or upregulates
expression of SDF-1 can comprise natural or synthetic nucleic acids
as described herein that are incorporated into recombinant nucleic
acid constructs, typically DNA constructs, capable of introduction
into and replication in the cells of the myocardial tissue. Such a
construct can include a replication system and sequences that are
capable of transcription and translation of a polypeptide-encoding
sequence in a given cell.
[0087] One method of introducing the agent into a target cell
involves using gene therapy. Gene therapy in some embodiments of
the application can be used to express SDF-1 protein from a cell of
the weakened region, ischemic region, and/or peri-infarct region of
the myocardial tissue in vivo.
[0088] In an aspect of the application, the gene therapy can use a
vector including a nucleotide encoding an SDF-1 protein. A "vector"
(sometimes referred to as gene delivery or gene transfer "vehicle")
refers to a macromolecule or complex of molecules comprising a
polynucleotide to be delivered to a target cell, either in vitro or
in vivo. The polynucleotide to be delivered may comprise a coding
sequence of interest in gene therapy. Vectors include, for example,
viral vectors (such as adenoviruses (`Ad`), adeno-associated
viruses (AAV), and retroviruses), non-viral vectors, liposomes, and
other lipid-containing complexes, and other macromolecular
complexes capable of mediating delivery of a polynucleotide to a
target cell.
[0089] Vectors can also comprise other components or
functionalities that further modulate gene delivery and/or gene
expression, or that otherwise provide beneficial properties to the
targeted cells. Such other components include, for example,
components that influence binding or targeting to cells (including
components that mediate cell-type or tissue-specific binding);
components that influence uptake of the vector nucleic acid by the
cell; components that influence localization of the polynucleotide
within the cell after uptake (such as agents mediating nuclear
localization); and components that influence expression of the
polynucleotide. Such components also might include markers, such as
detectable and/or selectable markers that can be used to detect or
select for cells that have taken up and are expressing the nucleic
acid delivered by the vector. Such components can be provided as a
natural feature of the vector (such as the use of certain viral
vectors which have components or functionalities mediating binding
and uptake), or vectors can be modified to provide such
functionalities.
[0090] Selectable markers can be positive, negative or
bifunctional. Positive selectable markers allow selection for cells
carrying the marker, whereas negative selectable markers allow
cells carrying the marker to be selectively eliminated. A variety
of such marker genes have been described, including bifunctional
(i.e., positive/negative) markers (see, e.g., Lupton, S., WO
92/08796, published May 29, 1992; and Lupton, S., WO 94/28143,
published Dec. 8, 1994). Such marker genes can provide an added
measure of control that can be advantageous in gene therapy
contexts. A large variety of such vectors are known in the art and
are generally available.
[0091] Vectors for use herein include viral vectors, lipid based
vectors and other non-viral vectors that are capable of delivering
a nucleotide to the cells of weakened region, ischemic region,
and/or peri-infarct region of the myocardial tissue. The vector can
be a targeted vector, especially a targeted vector that
preferentially binds to the cells of weakened region, ischemic
region, and/or peri-infarct region of the myocardial tissue. Viral
vectors for use in the methods herein can include those that
exhibit low toxicity to the cells of weakened region, ischemic
region, and/or peri-infarct region of the myocardial tissue and
induce production of therapeutically useful quantities of SDF-1
protein in a tissue-specific manner.
[0092] Examples of viral vectors are those derived from adenovirus
(Ad) or adeno-associated virus (AAV). Both human and non-human
viral vectors can be used and the recombinant viral vector can be
replication-defective in humans. Where the vector is an adenovirus,
the vector can comprise a polynucleotide having a promoter operably
linked to a gene encoding the SDF-1 protein and is
replication-defective in humans.
[0093] Other viral vectors that can be use in accordance with
method of the application include herpes simplex virus (HSV)-based
vectors. HSV vectors deleted of one or more immediate early genes
(IE) are advantageous because they are generally non-cytotoxic,
persist in a state similar to latency in the target cell, and
afford efficient target cell transduction. Recombinant HSV vectors
can incorporate approximately 30 kb of heterologous nucleic
acid.
[0094] Retroviruses, such as C-type retroviruses and lentiviruses,
might also be used in some embodiments of the application. For
example, retroviral vectors may be based on murine leukemia virus
(MLV). See, e.g., Hu and Pathak, Pharmacol. Rev. 52:493-511, 2000
and Fong et al., Crit. Rev. Ther. Drug Carrier Syst. 17:1-60, 2000.
MLV-based vectors may contain up to 8 kb of heterologous
(therapeutic) DNA in place of the viral genes. The heterologous DNA
may include a tissue-specific promoter and an SDF-1 nucleic acid.
In methods of delivery to cells proximate the wound, it may also
encode a ligand to a tissue specific receptor.
[0095] Additional retroviral vectors that might be used are
replication-defective lentivirus-based vectors, including human
immunodeficiency (HIV)-based vectors. See, e.g., Vigna and Naldini,
J. Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Virol.
72:8150-8157, 1998. Lentiviral vectors are advantageous in that
they are capable of infecting both actively dividing and
non-dividing cells. They are also highly efficient at transducing
human epithelial cells.
[0096] Lentiviral vectors for use in the methods herein may be
derived from human and non-human (including SIV) lentiviruses.
Examples of lentiviral vectors include nucleic acid sequences
required for vector propagation as well as a tissue-specific
promoter operably linked to a SDF-1 gene. These former may include
the viral LTRs, a primer binding site, a polypurine tract, att
sites, and an encapsidation site.
[0097] A lentiviral vector may be packaged into any suitable
lentiviral capsid. The substitution of one particle protein with
another from a different virus is referred to as "pseudotyping".
The vector capsid may contain viral envelope proteins from other
viruses, including murine leukemia virus (MLV) or vesicular
stomatitis virus (VSV). The use of the VSV G-protein yields a high
vector titer and results in greater stability of the vector virus
particles.
[0098] Alphavirus-based vectors, such as those made from semliki
forest virus (SFV) and sindbis virus (SIN) might also be used
herein. Use of alphaviruses is described in Lundstrom, K.,
Intervirology 43:247-257, 2000 and Perri et al., Journal of
Virology 74:9802-9807, 2000.
[0099] Recombinant, replication-defective alphavirus vectors are
advantageous because they are capable of high-level heterologous
(therapeutic) gene expression, and can infect a wide target cell
range. Alphavirus replicons may be targeted to specific cell types
by displaying on their virion surface a functional heterologous
ligand or binding domain that would allow selective binding to
target cells expressing a cognate binding partner. Alphavirus
replicons may establish latency, and therefore long-term
heterologous nucleic acid expression in a target cell. The
replicons may also exhibit transient heterologous nucleic acid
expression in the target cell.
[0100] In many of the viral vectors compatible with methods of the
application, more than one promoter can be included in the vector
to allow more than one heterologous gene to be expressed by the
vector. Further, the vector can comprise a sequence which encodes a
signal peptide or other moiety which facilitates the expression of
a SDF-1 gene product from the target cell.
[0101] To combine advantageous properties of two viral vector
systems, hybrid viral vectors may be used to deliver a SDF-1
nucleic acid to a target tissue. Standard techniques for the
construction of hybrid vectors are well-known to those skilled in
the art. Such techniques can be found, for example, in Sambrook, et
al., In Molecular Cloning: A laboratory manual. Cold Spring Harbor,
N.Y. or any number of laboratory manuals that discuss recombinant
DNA technology. Double-stranded AAV genomes in adenoviral capsids
containing a combination of AAV and adenoviral ITRs may be used to
transduce cells. In another variation, an AAV vector may be placed
into a "gutless", "helper-dependent" or "high-capacity" adenoviral
vector. Adenovirus/AAV hybrid vectors are discussed in Lieber et
al., J. Virol. 73:9314-9324, 1999. Retrovirus/adenovirus hybrid
vectors are discussed in Zheng et al., Nature Biotechnol.
18:176-186, 2000. Retroviral genomes contained within an adenovirus
may integrate within the target cell genome and effect stable SDF-1
gene expression.
[0102] Other nucleotide sequence elements which facilitate
expression of the SDF-1 gene and cloning of the vector are further
contemplated. For example, the presence of enhancers upstream of
the promoter or terminators downstream of the coding region, for
example, can facilitate expression.
[0103] In accordance with another aspect of the application, a
tissue-specific promoter, can be fused to a SDF-1 gene. By fusing
such tissue specific promoter within the adenoviral construct,
transgene expression is limited to a particular tissue. The
efficacy of gene expression and degree of specificity provided by
tissue specific promoters can be determined, using the recombinant
adenoviral system described herein.
[0104] In addition to viral vector-based methods, non-viral methods
may also be used to introduce a SDF-1 nucleic acid into a target
cell. A review of non-viral methods of gene delivery is provided in
Nishikawa and Huang, Human Gene Ther. 12:861-870, 2001. An example
of a non-viral gene delivery method according to the invention
employs plasmid DNA to introduce a SDF-1 nucleic acid into a cell.
Plasmid-based gene delivery methods are generally known in the art.
In one example, the plasmid vector can have a structure as shown
schematically in FIG. 7. The plasmid vector of FIG. 7 includes a
CMV enhancer and CMV promoter upstream of an SDF-1.alpha. cDNA
(RNA) sequence.
[0105] Optionally, a synthetic gene transfer molecules can be
designed to form multimolecular aggregates with plasmid SDF-1 DNA.
These aggregates can be designed to bind to cells of weakened
region, ischemic region, and/or peri-infarct region of the
myocardial tissue. Cationic amphiphiles, including lipopolyamines
and cationic lipids, may be used to provide receptor-independent
SDF-1 nucleic acid transfer into target cells (e.g.,
cardiomyocytes). In addition, preformed cationic liposomes or
cationic lipids may be mixed with plasmid DNA to generate
cell-transfecting complexes. Methods involving cationic lipid
formulations are reviewed in Felgner et al., Ann N.Y. Acad. Sci.
772:126-139, 1995 and Lasic and Templeton, Adv. Drug Delivery Rev.
20:221-266, 1996. For gene delivery, DNA may also be coupled to an
amphipathic cationic peptide (Fominaya et al., J. Gene Med.
2:455-464, 2000).
[0106] Methods that involve both viral and non-viral based
components may be used herein. For example, an Epstein Barr virus
(EBV)-based plasmid for therapeutic gene delivery is described in
Cui et al., Gene Therapy 8:1508-1513, 2001. Additionally, a method
involving a DNA/ligand/polycationic adjunct coupled to an
adenovirus is described in Curiel, D. T., Nat. Immun. 13:141-164,
1994.
[0107] Additionally, the SDF-1 nucleic acid can be introduced into
the target cell by transfecting the target cells using
electroporation techniques. Electroporation techniques are well
known and can be used to facilitate transfection of cells using
plasmid DNA.
[0108] Vectors that encode the expression of SDF-1 can be delivered
to the target cell in the form of an injectable preparation
containing pharmaceutically acceptable carrier, such as saline, as
necessary. Other pharmaceutical carriers, formulations and dosages
can also be used in accordance with the present invention.
[0109] In one aspect of the invention, the vector can comprise an
SDF-1 plasmid, such as for example in FIG. 7. SDF-1 plasmid can be
delivered to cells of the weakened region, ischemic region, and/or
peri-infarct region of the myocardial tissue by direct injection of
the SDF-1 plasmid vector into the weakened region, ischemic region,
and/or peri-infarct region of the myocardial tissue at an amount
effective to improve at least one myocardial functional parameters,
such as left ventricular volume, left ventricular area, left
ventricular dimension, or cardiac function as well as improve the
subject's 6-minute walk test (6MWT) or New York Heart Association
(NYHA) functional classification. By injecting the vector directly
into or about the periphery of the weakened region, ischemic
region, and/or peri-infarct region of the myocardial tissue, it is
possible to target the vector transfection rather effectively, and
to minimize loss of the recombinant vectors. This type of injection
enables local transfection of a desired number of cells, especially
about the weakened region, ischemic region, and/or peri-infarct
region of the myocardial tissue, thereby maximizing therapeutic
efficacy of gene transfer, and minimizing the possibility of an
inflammatory response to viral proteins.
[0110] In an aspect of the application, the SDF-1 plasmid can be
administered to the weakened, ischemic, and/or peri-infarct region
in multiple injections of a solution of SDF-1 expressing plasmid
DNA with each injection comprising about 0.33 mg/ml to about 5
mg/ml of SDF-1 plasmid/solution. In one example, the SDF-1 plasmid
can be administered to the weakened, ischemic, and/or peri-infarct
region in at least about 10 injections, at least about 15
injections, or at least about 20 injections. Multiple injections of
the SDF-1 plasmid to the weakened, ischemic, and/or peri-infarct
region allows a greater area and/or number of cells of the
weakened, ischemic, and/or peri-infarct region to be treated.
[0111] Each injection administered to the weakened, ischemic,
and/or peri-infarct region can have a volume of at least about 0.2
ml. The total volume of solution that includes the amount of SDF-1
plasmid administered to the weakened, ischemic, and/or peri-infarct
region that can improve at least one functional parameter of the
heart is at least about 10 ml.
[0112] In one example, the SDF-1 plasmid can be administered to the
weakened, ischemic, and/or peri-infarct region in at least about 10
injections. Each injection administered to the weakened, ischemic,
and/or peri-infarct region can have a volume of at least about 0.2
ml. The SDF-1 can be expressed in the weakened, ischemic, and/or
peri-infarct region for greater than about three days.
[0113] For example, each injection of solution including SDF-1
expressing plasmid can have an injection volume of at least about
0.2 ml and an SDF-1 plasmid concentration per injection of about
0.33 mg/ml to about 5 mg/ml. In another aspect of the application,
at least one functional parameter of the of the heart can be
improved by injecting the SDF-1 plasmid into the weakened,
ischemic, and/or peri-infarct region of the heart at an injection
volume per site of at least about 0.2 ml, in at least about 10
injection sites, and at an SDF-1 plasmid concentration per
injection of about 0.33 mg/ml to about 5 mg/ml.
[0114] It was found in a porcine model of congestive heart failure
that injections of a solution of SDF-1 plasmid having concentration
of less about 0.33 mg/ml or greater than about 5 mg/ml and an
injection volume per injection site less than about 0.2 ml to a
porcine model of heart failure resulted in little if any functional
improvement of the left ventricular volume, left ventricular area,
left ventricular dimension, or cardiac function of the treated
heart.
[0115] In another aspect of the application, the amount of SDF-1
plasmid administered to the weakened, ischemic, and/or peri-infarct
region that can improve at least one functional parameter of the
heart is greater than about 4 mg and less than about 100 mg per
therapeutic intervention. The amount of SDF-1 plasmid administered
by therapeutic intervention herein refers to the total SDF-1
plasmid administered to the subject during a therapeutic procedure
designed to affect or elicit a therapeutic effect. This can include
the total SDF-1 plasmid administered in single injection for a
particular therapeutic intervention or the total SDF-1 plasmid that
is administered by multiple injections for a therapeutic
intervention. It was found in a porcine model of congestive heart
failure that administration of about 4 mg SDF-1 plasmid DNA via
direct injection of the SDF-1 plasmid to the heart resulted in no
functional improvement of the left ventricular volume, left
ventricular area, left ventricular dimension, or cardiac function
of the treated heart. Moreover, administration of about 100 mg of
SDF-1 plasmid DNA via direct injection of the SDF-1 plasmid to the
heart resulted in no functional improvement of the left ventricular
volume, left ventricular area, left ventricular dimension, or
cardiac function of the treated heart.
[0116] In some aspects of the application, the SDF-1 can be
expressed at a therapeutically effective amount or dose in the
weakened, ischemic, and/or peri-infarct region after transfection
with the SDF-1 plasmid vector for greater than about three days.
Expression of SDF-1 at a therapeutically effective dose or amount
for greater three days can provide a therapeutic effect to
weakened, ischemic, and/or peri-infarct region. Advantageously, the
SDF-1 can be expressed in the weakened, ischemic, and/or
peri-infarct region after transfection with the SDF-1 plasmid
vector at a therapeutically effective amount for less than about 90
days to mitigate potentially chronic and/or cytotoxic effects that
may inhibit the therapeutic efficacy of the administration of the
SDF-1 to the subject.
[0117] It will be appreciated that the amount, volume,
concentration, and/or dosage of SDF-1 plasmid that is administered
to any one animal or human depends on many factors, including the
subject's size, body surface area, age, the particular composition
to be administered, sex, time and route of administration, general
health, and other drugs being administered concurrently. Specific
variations of the above noted amounts, volumes, concentrations,
and/or dosages of SDF-1 plasmid can be readily be determined by one
skilled in the art using the experimental methods described
below.
[0118] In another aspect of the application, the SDF-1 plasmid can
be administered by direct injection using catheterization, such as
endo-ventricular catheterization or intra-myocardial
catheterization. In one example, a deflectable guide catheter
device can be advanced to a left ventricle retrograde across the
aortic valve. Once the device is positioned in the left ventricle,
SDF-1 plasmid can be injected into the peri-infarct region (both
septal and lateral aspect) area of the left ventricle. Typically,
1.0 ml of SDF-1 plasmid solution can be injection over a period of
time of about 60 seconds. The subject be treated can receive at
least about 10 injection (e.g., about 15 to about 20 injections in
total).
[0119] The myocardial tissue of the subject can be imaged prior to
administration of the SDF-1 plasmid to define the area of weakened,
ischemic, and/or peri-infarct region prior to administration of the
SDF-1 plasmid. Defining the weakened, ischemic, and/or peri-infarct
region by imaging allows for more accurate intervention and
targeting of the SDF-1 plasmid to the weakened, ischemic, and/or
peri-infarct region. The imaging technique used to define the
weakened, ischemic, and/or peri-infarct region of the myocardial
tissue can include any known cardio-imaging technique. Such imaging
techniques can include, for example, at least one of
echocardiography, magnetic resonance imaging, coronary angiogram,
electroanatomical mapping, or fluoroscopy. It will be appreciated
that other imaging techniques that can define the weakened,
ischemic, and/or peri-infarct region can also be used.
[0120] Optionally, other agents besides SDF-1 nucleic acids (e.g.,
SDF-1 plasmids) can be introduced into the weakened, ischemic,
and/or peri-infarct region of the myocardial tissue to promote
expression of SDF-1 from cells of the weakened, ischemic, and/or
peri-infarct region. For example, agents that increase the
transcription of a gene encoding SDF-1 increase the translation of
an mRNA encoding SDF-1, and/or those that decrease the degradation
of an mRNA encoding SDF-1 could be used to increase SDF-1 protein
levels. Increasing the rate of transcription from a gene within a
cell can be accomplished by introducing an exogenous promoter
upstream of the gene encoding SDF-1. Enhancer elements, which
facilitate expression of a heterologous gene, may also be
employed.
[0121] Other agents can include other proteins, chemokines, and
cytokines, that when administered to the target cells can
upregulate expression SDF-1 form the weakened, ischemic, and/or
peri-infarct region of the myocardial tissue. Such agents can
include, for example: insulin-like growth factor (IGF)-1, which was
shown to upregulate expression of SDF-1 when administered to
mesenchymal stem cells (MSCs) (Circ. Res. 2008, November 21;
103(11):1300-98); sonic hedgehog (Shh), which was shown to
upregulate expression of SDF-1 when administered to adult
fibroblasts (Nature Medicine, Volume 11, Number 11, Nov. 23);
transforming growth factor .beta. (TGF-.beta.); which was shown to
upregulate expression of SDF-1 when administered to human
peritoneal mesothelial cells (HPMCs); IL-1.beta., PDGF, VEGF,
TNF-.alpha., and PTH, which are shown to upregulate expression of
SDF-1, when administered to primary human osteoblasts (HOBs) mixed
marrow stromal cells (BMSCs), and human osteoblast-like cell lines
(Bone, 2006, April; 38(4): 497-508); thymosin .beta.4, which was
shown to upregulate expression when administered to bone marrow
cells (BMCs) (Curr. Pharm. Des. 2007; 13(31):3245-51; and hypoxia
inducible factor 1.alpha. (HIF-1), which was shown to upregulate
expression of SDF-1 when administered to bone marrow derived
progenitor cells (Cardiovasc. Res. 2008, E. Pub.). These agents can
be used to treat specific cardiomyopathies where such cells capable
of upregulating expression of SDF-1 with respect to the specific
cytokine are present or administered.
[0122] The SDF-1 protein or agent, which causes increases, and/or
upregulates expression of SDF-1, can be administered to the
weakened, ischemic, and/or peri-infarct region of the myocardial
tissue neat or in a pharmaceutical composition. The pharmaceutical
composition can provide localized release of the SDF-1 or agent to
the cells of the weakened, ischemic, and/or peri-infarct region
being treated. Pharmaceutical compositions in accordance with the
application will generally include an amount of SDF-1 or agent
admixed with an acceptable pharmaceutical diluent or excipient,
such as a sterile aqueous solution, to give a range of final
concentrations, depending on the intended use. The techniques of
preparation are generally well known in the art as exemplified by
Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing
Company, 1980, incorporated herein by reference. Moreover, for
human administration, preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biological Standards.
[0123] The pharmaceutical composition can be in a unit dosage
injectable form (e.g., solution, suspension, and/or emulsion).
Examples of pharmaceutical formulations that can be used for
injection include sterile aqueous solutions or dispersions and
sterile powders for reconstitution into sterile injectable
solutions or dispersions. The carrier can be a solvent or
dispersing medium containing, for example, water, ethanol, polyol
(e.g., glycerol, propylene glycol, liquid polyethylene glycol, and
the like), dextrose, saline, or phosphate-buffered saline, suitable
mixtures thereof and vegetable oils.
[0124] Proper fluidity can be maintained, for example, by the use
of a coating, such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil,
olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and
esters, such as isopropyl myristate, may also be used as solvent
systems for compound compositions.
[0125] Additionally, various additives, which enhance the
stability, sterility, and isotonicity of the compositions,
including antimicrobial preservatives, antioxidants, chelating
agents, and buffers, can be added. Prevention of the action of
microorganisms can be ensured by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, and the like. In many cases, it will be desirable to
include isotonic agents, for example, sugars, sodium chloride, and
the like. Prolonged absorption of the injectable pharmaceutical
form can be brought about by the use of agents delaying absorption,
for example, aluminum monostearate and gelatin. According to
methods described herein, however, any vehicle, diluent, or
additive used would have to be compatible with the compounds.
[0126] Sterile injectable solutions can be prepared by
incorporating the compounds utilized in practicing the methods
described herein in the required amount of the appropriate solvent
with various amounts of the other ingredients, as desired.
[0127] Pharmaceutical "slow release" capsules or "sustained
release" compositions or preparations may be used and are generally
applicable. Slow release formulations are generally designed to
give a constant drug level over an extended period and may be used
to deliver the SDF-1 or agent. The slow release formulations are
typically implanted in the vicinity of the weakened, ischemic,
and/or peri-infarct region of the myocardial tissue.
[0128] Examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
SDF-1 or agent, which matrices are in the form of shaped articles,
e.g., films or microcapsule. Examples of sustained-release matrices
include polyesters; hydrogels, for example,
poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol);
polylactides, e.g., U.S. Pat. No. 3,773,919; copolymers of
L-glutamic acid and y ethyl-L-glutamate; non-degradable
ethylene-vinyl acetate; degradable lactic acid-glycolic acid
copolymers, such as the LUPRON DEPOT (injectable microspheres
composed of lactic acid-glycolic acid copolymer and leuprolide
acetate); and poly-D-(-)-3-hydroxybutyric acid.
[0129] While polymers, such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated, SDF-1 or the agent can remain in the body for a long
time, and may denature or aggregate as a result of exposure to
moisture at 37.degree. C., thus reducing biological activity and/or
changing immunogenicity. Rational strategies are available for
stabilization depending on the mechanism involved. For example, if
the aggregation mechanism involves intermolecular S--S bond
formation through thio-disulfide interchange, stabilization is
achieved by modifying sulfhydryl residues, lyophilizing from acidic
solutions, controlling moisture content, using appropriate
additives, developing specific polymer matrix compositions, and the
like.
[0130] In certain embodiments, liposomes and/or nanoparticles may
also be employed with the SDF-1 or agent. The formation and use of
liposomes is generally known to those of skill in the art, as
summarized below.
[0131] Liposomes are formed from phospholipids that are dispersed
in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 {acute
over (.ANG.)}, containing an aqueous solution in the core.
[0132] Phospholipids can form a variety of structures other than
liposomes when dispersed in water, depending on the molar ratio of
lipid to water. At low ratios, the liposome is the preferred
structure. The physical characteristics of liposomes depend on pH,
ionic strength and the presence of divalent cations. Liposomes can
show low permeability to ionic and polar substances, but at
elevated temperatures undergo a phase transition which markedly
alters their permeability. The phase transition involves a change
from a closely packed, ordered structure, known as the gel state,
to a loosely packed, less-ordered structure, known as the fluid
state. This occurs at a characteristic phase-transition temperature
and results in an increase in permeability to ions, sugars and
drugs.
[0133] Liposomes interact with cells via four different mechanisms:
Endocytosis by phagocytic cells of the reticuloendothelial system
such as macrophages and neutrophils; adsorption to the cell
surface, either by nonspecific weak hydrophobic or electrostatic
forces, or by specific interactions with cell-surface components;
fusion with the plasma cell membrane by insertion of the lipid
bilayer of the liposome into the plasma membrane, with simultaneous
release of liposomal contents into the cytoplasm; and by transfer
of liposomal lipids to cellular or subcellular membranes, or vice
versa, without any association of the liposome contents. Varying
the liposome formulation can alter which mechanism is operative,
although more than one may operate at the same time.
[0134] Nanocapsules can generally entrap compounds in a stable and
reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) should be designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use in the methods, and
such particles may be are easily made.
[0135] For preparing pharmaceutical compositions from the compounds
of the application, pharmaceutically acceptable carriers can be in
any form (e.g., solids, liquids, gels, etc.). A solid carrier can
be one or more substances, which may also act as diluents,
flavoring agents, binders, preservatives, and/or an encapsulating
material. The following examples are for the purpose of
illustration only and are not intended to limit the scope of the
claims, which are appended hereto.
EXAMPLES
Example 1
[0136] Stromal cell-derived factor-1 or SDF-1 is a
naturally-occurring chemokine whose expression is rapidly
upregulated in response to tissue injury. SDF-1 induction
stimulates a number of protective anti-inflammatory pathways,
causes the down regulation of pro-inflammatory mediators (such as
MMP-9 and IL-8), and can protect cells from apoptosis. Furthermore,
SDF-1 is a strong chemoattractant of organ specific and bone marrow
derived stem cells and progenitor cells to the site of tissue
damage, which promotes tissue preservation and blood vessel
development. Based on observations that increased expression of
SDF-1 led to improved cardiac function in ischemic animal models,
we focused on developing a non-viral, naked-DNA SDF-1-encoding
plasmid for treatment of ischemic cardiovascular disease. During
the course of development, the plasmid was optimized based on cell
culture and small animal study results described below. The plasmid
ACL-01110Sk was selected based on its ability to express transgenes
in cardiac tissue and to consistently improve cardiac function in
pre-clinical animal models of ischemic cardiomyopathy. SDF-1
transgene expression in ACL-01110Sk is driven by the CMV
enhancer/promoter, CMV-intron A, and the RU5 translational
enhancer. The drug product, JVS-100 (formerly ACRX-100), is
composed of plasmid ACL-01110Sk in 5% dextrose.
[0137] Initial studies in a rat model of heart failure demonstrated
that ACL-01110S (an SDF-1 expressing precursor to ACL-01110Sk)
improved cardiac function after injection of the plasmid directly
into the infarct border zone of the rat hearts four weeks following
an MI. Benefits were sustained for at least 8-10 weeks
post-injection and correlated with increased vasculogenesis in the
ACL-01110S treated animals. ACL-01110S was modified to optimize its
expression profile.
Plasmid Dose-Dependent Expression in a Rat Model of MI
[0138] To determine the plasmid dose per injection that would
provide maximal expression in rat cardiac tissue, escalating doses
(10, 50, 100, 500 .mu.g) of the ACL-00011L luciferase plasmid were
injected into infarcted rat hearts. Lewis rats were subjected to a
median sternotomy and the left anterior descending artery (LAD) was
permanently ligated, and injected peri-MI at one site with 100
.mu.l ACL-00011L plasmid in PBS. Whole body luciferase expression
was measured in each dose cohort (n=3) by non-invasive
bioluminescent imaging (Xenogen, Hopkinton, Mass.) at baseline and
at 1, 2, 3, 4, and 5 days post-injection. The peak expression
increased up to a dose of 100 .mu.g and saturated at higher doses.
Based on this dose-response curve, a dose of 100 .mu.g was
determined to be sufficient for maximal plasmid expression in rat
hearts. ACL-00011L expressed the luciferase gene from a vector
backbone equivalent to that used in construction of ACL-00011S,
which expresses SDF-1.
Comparison of Cardiac Vector Expression in a Rat Model of Ischemic
Heart Failure
[0139] The luciferase expressing equivalents of several SDF-1
plasmid candidates were tested for expression in cardiac tissue in
a rat model of myocardial infarct (MI). Plasmid candidates differed
in the promoters driving expression and presence of enhancer
elements. Lewis rats were subjected to a median sternotomy and the
left anterior descending artery (LAD) was permanently ligated and
the chest was closed. Four weeks later, the chest was reopened, and
the luciferase expressing plasmids was directly injected (100 .mu.g
in 100 .mu.l per injection) into 4 peri-Myocardial infarction
sites. At 1, 2, 4, 6, 8, and 10 days post-injection (and every 3-4
days following), rats were anesthetized, injected with luciferin
and imaged with a whole-body Xenogen Luciferase imaging system.
[0140] The two CMV driven plasmids tested, ACL-00011L and
ACL-01110L yielded detectable luciferase expression within 24 hours
of injection with an initial peak of expression at 2 days
post-injection.
[0141] ACL-01110L peak expression was 7 times greater than
ACL-00011L and expression was approximately 10 days longer (lasting
up to 16 days post injection). In contrast, ACL-00021L (.alpha.MHC
driven plasmid) showed no initial peak, but expressed at a
low-level through day 25 post-injection. These results support
previous studies demonstrating that CMV driven plasmids can be used
for localized, transient protein expression in the heart and that
the timeframe of therapeutic protein expression can be modulated
through the inclusion of enhancer elements.
Efficacy of SDF-1 Plasmids in Rat Model of MI
[0142] SDF-1-encoding plasmids were tested in a rat model of MI to
determine if functional cardiac benefit could be achieved. Lewis
rats were subjected to a median sternotomy and the LAD was
permanently ligated immediately distal to the first bifurcation.
Four weeks later, the chest was reopened, and one of three SDF-1
expressing plasmids (ACL-01110S, ACL-00011S, or ACL-00021S) or
saline was injected (100 .mu.g per 100 .mu.l injection) into 4
peri-MI sites:
[0143] At baseline (pre-injection), and 2, 4, and 8 weeks
post-injection, rats were anesthetized and imaged with M-mode
echocardiography. LVEF, fractional shortening, and LV dimensions
were measured by a trained sonographer who was blinded to
randomization.
[0144] A strong trend in improvement in cardiac function was
observed with both CMV driven plasmids, ACL-01110S and ACL-00011S,
compared to saline controls. ACL-01110S elicited a statistically
significant increase in fractional shortening at four weeks that
was sustained 8 weeks after injection. In contrast, no difference
in function was observed between .alpha.MHC driven plasmid
ACL-000215 and saline. Furthermore, compared to control, the
ACL-01110S and the ACL-00011S-treated animals had significant
increases in large vessel density (ACL-01110S: 21.+-.1.8
vessels/mm.sup.2; ACL-000115: 17.+-.1.5 vessels/mm.sup.2; saline:
6.+-.0.7 vessels/mm.sup.2, p<0.001 for both vs. saline) and
reduced infarct size (ACL-01110S: 16.9.+-.2.8%; ACL-000115:
17.8.+-.2.6%; saline: 23.8.+-.4.5%). Importantly, treatment with
ACL-01110S demonstrated the largest improvement in cardiac function
and vasculogenesis, and caused the largest reduction in infarct
size.
[0145] In summary, in a rat model of ischemic heart failure, both
SDF-1-encoding plasmids driven by a CMV promoter provided
functional cardiac benefit, increased vasculogenesis, and reduction
in infarct size compared to saline treatment. In all parameters
tested, ACL-01110S provided the most significant benefit.
Transfection Efficiency of ACL-01110Sk and ACl-01010Sk in H9C2
Cells
[0146] In vitro transfection of H9C2 myocardial cells without
transfection reagents (i.e.,--naked plasmid DNA was added to cells
in culture) were used to estimate in vivo transfection efficiencies
of GFP versions of Juventas lead plasmid vectors, ACL-01110Sk and
ACL-01010Sk. H9C2 cells were cultured in vitro and various amounts
of pDNA (0.5 .mu.g, 2.0 .mu.g, 4.0 .mu.g, 5.0 .mu.g) were added in
5% dextrose. The GFP vectors were constructed from the ACL-01110Sk
(ACL-01110G) or ACL-01010Sk (ACL-01010G) backbones. At Day 3
post-transfection, GFP fluorescence was assessed by FACS to
estimate transfection efficiency. The transfection efficiencies for
the ACL-01110G and ACL-01010G vectors in 5% dextrose ranged from
1.08-3.01%. At each amount of pDNA tested, both vectors had similar
in vitro transfection efficiencies. We conclude that the 1-3%
transfection efficiency observed in this study is in line with
findings from previous studies demonstrating a similar level of in
vivo transfection efficiency. Specifically, JVS-100 will transfect
a limited but sufficient number of cardiac cells to produce
therapeutic amounts of SDF-1.
Example 2
Expression of Plasmid in Porcine Myocardium
[0147] A porcine occlusion/reperfusion MI model of the left
anterior descending artery (LAD) was selected as an appropriate
large animal model to test the efficacy and safety of ACRX-100. In
this model, 4 weeks recovery is given between MI and treatment to
allow time for additional cardiac remodeling and to simulate
chronic ischemic heart failure.
Surgical Procedure
[0148] Yorkshire pigs were anesthesitzed and heparanized to an
activated clotting time (ACT) of .gtoreq.300 seconds, and
positioned in dorsal recumbency. To determine the contour of the
LV, left ventriculography was performed in both the
Anterior-Posterior and Lateral views.
Delivery of Luciferase Plasmid into Porcine Myocardium
[0149] A deflectable guide catheter device was advanced to the left
ventricle retrograde across the aortic valve, the guide wire was
removed, and an LV endocardial needle injection catheter was
entered through the guide catheter into the LV cavity. Luciferase
plasmid was injected at 4 sites at a given volume and concentration
were made into either the septal or lateral wall of the heart. Five
combinations of plasmid concentration (0.5, 2, or 4 mg/ml) and site
injection volumes (0.2, 0.5, 1.0 ml) were tested. Plasmid at 0.5
mg/ml was buffered in USP Dextrose, all others were buffered in USP
Phosphate Buffered Saline. For each injection, the needle was
inserted into the endocardium, and the gene solution was injected
at a rate of 0.8-1.5 ml/minute. Following injection, the needle was
held in place for 15 seconds and then withdrawn. After injections
were completed, all instrumentation was removed, the incision was
closed, and the animal was allowed to recover.
Harvesting of Myocardial Tissue
[0150] On Day 3 post injection, the animals were submitted to
necropsy. Following euthanasia, the heart was removed, weighed, and
perfused with Lactate Ringers Solution until clear of blood. The LV
was opened and the injection sites identified. A 1 cm square cube
of tissue was taken around each injection site. Four (4) cubes
harvested from the posterior wall remote from any injection sites
served as negative controls. The tissue samples were frozen in
liquid nitrogen and stored at -20 to -70.degree. C.
Assessment of Luciferase Expression
[0151] The tissue samples were thawed and placed in a 5 ml glass
tube. Lysis buffer (0.5-1.0 ml) was added and tissue was disrupted
using Polytron homogenization (model PT1200) on ice. Tissue
homogenate was centrifuged and protein concentration of the
supernatant was determined for each tissue sample using the Bio-rad
Detergent-Compatible (DC) protein assay and a standard curve of
known amounts of bovine serum albumin (BSA). Tissue sample
homogenate (1-10 .mu.l) was assayed using the Luciferase assay kit
(Promega).
[0152] The results of the experiment are shown in FIG. 1. The data
shows that expression of the vector increases with increasing
injection volume and increasing concentration of DNA.
Example 3
Improvement in Cardiac Function by SDF-1 Plasmid Treatment in
Porcine Model of Ischemic Cardiomyopathy
Induction of Myocardial Infarction
[0153] Yorkshire pigs were anesthesitzed and heparanized to an
activated clotting time (ACT) of .gtoreq.250 seconds, and
positioned in dorsal recumbency. A balloon catheter was introduced
by advancing it through a guide catheter to the LAD to below the
first major bifurcation of the LAD. The balloon was then inflated
to a pressure sufficient to ensure complete occlusion of the
artery, and left inflated in the artery for 90-120 minutes.
Complete balloon inflation and deflation was verified with
fluoroscopy. The balloon was then removed, the incision was closed,
and the animal was allowed to recover.
Enrollment Criteria
[0154] One month post-MI, cardiac function in each pig was assessed
by echocardiography. If the LVEF was less than 40% and the LVESV
was greater than 56.7 ml, the pig was enrolled in the study.
Surgical Procedure
[0155] Each enrolled pig was anesthesitzed and heparanized to an
activated clotting time (ACT) of .gtoreq.300 seconds, and
positioned in dorsal recumbency. To determine the contour of the
LV, left ventriculography was performed in both the
Anterior-Posterior and Lateral views.
Delivery of SDF-1 Plasmid (ACL-01110Sk) Into Myocardium
[0156] Each pig was randomized to one of 3 sacrifice points: 3
days, 30 days, or 90 days post-treatment, and to one of four
treatment groups: control (20 injections, buffer only), low (15
injections, 0.5 mg/ml), mid (15 injections, 2.0 mg/ml), or high (20
injections, 5.0 mg/ml). All plasmid was buffered in USP Dextrose.
The injection procedure is described below.
[0157] A deflectable guide catheter device was advanced to the left
ventricle retrograde across the aortic valve, the guide wire was
removed, and an LV endocardial needle injection catheter was
entered through the guide catheter into the LV cavity. SDF-1
plasmid or buffer at randomized dose was loaded into 1 ml syringes
that were connected to the catheter. Each injection volume was 1.0
ml. For each injection, the needle was inserted into the
endocardium, and the solution was injected over 60 seconds.
Following injection, the needle was held in place for 15 seconds
and then withdrawn. After injections were completed, all
instrumentation was removed, the incision was closed, and the
animal was allowed to recover.
[0158] At sacrifice, samples of tissues from the heart and other
major organs were excised and flash frozen for PCR and
histopathological analysis.
Assessment of Cardiac Function
[0159] Each animal had cardiac function assessed by standard
2-dimensional echocardiography at day 0, 30, 60, and 90
post-injection (or until sacrifice). Measurements of left
ventricular volume, area, and wall motion score were made by an
independent core laboratory. The efficacy parameters measured are
shown below in Table 1.
TABLE-US-00002 TABLE 1 Echocardiographic Parameters Variable Name
Definition LVESV End Systolic Volume measured in parasternal
long-axis view LVEDV End Diastolic Volume measured in parasternal
long-axis view LVEF (LVEDV - LVESV)/LVEDV * 100% WMSI Average of
all readable wall motion scores based on ASE 17 segment model and
scoring system of 0-5.
[0160] The impact of SDF-1 plasmid on functional improvement is
shown in FIGS. 2-5. FIGS. 2-4 show that the low and mid doses of
SDF-1 plasmid improve LVESV, LVEF, and Wall Motion Score Index at
30 days post-injection compared to control; whereas, the high dose
does not show benefit. FIG. 5 demonstrates that the cardiac benefit
in the low and mid dose is sustained to 90 days, as both show a
marked attenuation in pathological remodeling, that is, a smaller
increase in LVESV, compared to control.
Assessment of Vasculogenesis
[0161] Animals that were sacrificed at 30 days were assessed for
vessel density in the left ventricle using 7 to 9 tissue samples
harvested from each formalin-fixed heart. Genomic DNA was extracted
and efficiently purified from formalin fixed tissue sample using a
mini-column purification procedure (Qiagen). Samples from SDF-1
treated and control animals were tested for presence of plasmid DNA
by quantitative PCR. Three to five tissue samples found to contain
copies of plasmid DNA at least 4-fold above background (except in
control animals) for each animal were used to prepare slides and
immunostained with isolectin. Cross-sections were identified and
vessels counted in 20-40 random fields per tissue. The vessels per
field were converted to vessels/mm.sup.2 and were averaged for each
animal. For each dose, data is reported as the average
vessels/mm.sup.2 from all animals receiving that dose.
[0162] FIG. 6 shows that both doses that provided functional
benefit also significantly increase vessel density at 30 days
compared to control. In contrast, the high dose, which did not
improve function, did not substantially increase vessel density.
This data provides a putative biologic mechanism by which SDF-1
plasmid is improving cardiac function in ischemic
cardiomyopathy.
Biodistribution Data
[0163] JVS-100 distribution in cardiac and non-cardiac tissues was
measured 3, 30 and 90 days after injection in the pivotal efficacy
and toxicology study in the pig model of MI. In cardiac tissue, at
each time point, average JVS-100 plasmid concentration increased
with dose. Art each dose, JVS-100 clearance was observed at 3, 30
and 90 days following injection with approximately 99.999999%
cleared from cardiac tissue at Day 90. JVS-100 was distributed to
non-cardiac organs with relatively high blood flow (e.g. heart,
kidney, liver, and lung) with the highest concentrations noted 3
days following injection. JVS-100 was present primarily in the
kidney, consistent with renal clearance of the plasmid. There were
low levels of persistence at 30 days and JVS-100 was essentially
undetectable in non-cardiac tissues at 90 days.
Conclusions
[0164] Treatment with JVS-100 resulted in significantly increased
blood vessel formation and improved heart function in pigs with
ischemic heart failure following a single endomyocardial injection
of 7.5 and 30 mg. The highest dose of JVS-100 tested (100 mg)
showed a trend in increased blood vessel formation but did not show
improved heart function. None of the doses of JVS-100 were
associated with signs of toxicity, adverse effects on clinical
pathology parameters or histopathology. JVS-100 was distributed
primarily to the heart with approximately 99.999999% cleared from
cardiac tissue at 90 days following treatment. JVS-100 was
distributed to non-cardiac organs with relatively high blood flow
(e.g., heart, kidney, liver, and lung) with the highest
concentrations in the kidneys 3 days following injection. JVS-100
was essentially undetectable in the body 90 days after injection
with only negligible amounts of the administered dose found in
non-cardiac tissues. Based on these findings the no observed
adverse effect level (NOAEL) for JVS-100 in the pig model of MI was
100 mg administered by endomyocardial injection.
Example 4
Porcine Exploratory Study: LUC Injections by Transarterial
Injection in Chronic MI Pigs
Methods
[0165] One pig with a previous LAD occlusion/reperfusion MI and an
EF>40%, was injected with ACL-01110Sk with a Transarterial
catheter. Two injections in the LAD and 2 in the LCX were performed
with an injection volume of 2.5 ml and a total injection time of
125-130 sec. One additional injection in the LCX of 3.0 ml with a
total injection time of 150 sec was performed with contrast mixed
with the plasmid.
Sacrifice and Tissue Collection
[0166] Three days following the injections, the animal was
euthanized. After euthanasia, the heart was removed, drained of
blood, placed on an ice cold cutting board and further dissected by
the necropsy technician or pathologist. The non-injected myocardium
from the septum was obtained via opening the right ventricle. The
right ventricle was trimmed from the heart and placed in cold
cardioplegia. New scalpel blades were used for each of the
sections.
[0167] Next, the left ventricle was opened and the entire left
ventricle was excised by slicing into 6 sections cutting from apex
to base. The LV was evenly divided into 3 slices. Following
excision, each section was able to lay flat. Each section (3 LV
sections, 1 RV section, and 1 pectoral muscle) was placed in
separate labeled containers with cold cardioplegia on wet ice, and
transported for luciferase analysis.
Luciferase Imaging
[0168] All collected tissues were immersed in luciferin and imaged
with a Xenogen imaging system to determine plasmid expression.
Results
[0169] A representative image of the heart is shown in FIG. 8. The
colored spots denote areas of luciferase expression. These spots
showed Relative Light Units (RLUs) of greater than 10.sup.6 units,
more than 2 orders of magnitude above background. This data
demonstrated that the catheter delivered plasmid sufficient to
generate substantial plasmid expression over a significant portion
of the heart.
Example 5
Clinical Study Example
[0170] Ascending doses of JVS-100 are administered to treat HF in
subjects with ischemic cardiomyopathy. Safety is tracked at each
dose by documenting all adverse events (AEs), with the primary
safety endpoint being the number of major cardiac AEs at 30 days.
In each cohort, subjects will receive a single dose of JVS-100. In
all cohorts, therapy efficacy is evaluated by measuring the impact
on cardiac function via standard echocardiography measurements,
cardiac perfusion via Single Photon Emission Computed Tomography
(SPECT) imaging, New York Heart Association (NYHA) class, six
minute walk distance, and quality of life.
[0171] All subjects have a known history of systolic dysfunction,
prior MI, and no current cancer verified by up to date age
appropriate cancer screening. All subjects are screened with a
physician visit, and a cardiac echocardiogram. Further baseline
testing such as SPECT perfusion imaging, is performed. Each subject
receives fifteen (15) 1 ml injections of JVS-100 delivered by an
endocardial needle catheter to sites within the infarct border
zone. Three cohorts (A, B, C) will be studied. As shown in Table 2,
dose will be escalated by increasing the amount of DNA per
injection site while holding number of injection sites constant at
15 and injection volume at 1 ml. Subjects are monitored for
approximately 18 hours post-injection and have scheduled visits at
3 and 7 days post-injection to ensure that there are no safety
concerns. The patient remains in the hospital for 18 hours after
the injection to ensure all required blood collections (i.e.,
cardiac enzymes, plasma SDF-1 protein levels) are performed. All
subjects have follow-up at 30 days (1 month), 120 days (4 months),
and 360 days (12 months) to assess safety and cardiac function. The
primary safety endpoint are major adverse cardiac events (MACE)
within 1 month post-therapy delivery. AEs will be tracked for each
subject throughout the study. The following safety and efficacy
endpoints will be measured:
Safety:
[0172] Number of Major Adverse Cardiac Events (MACE) at 30 days
post-injection
[0173] Adverse Events throughout the 12 month follow-up period
[0174] Blood lab Analysis (Cardiac Enzymes, CBC, ANA)
[0175] SDF-1 Plasma Levels
[0176] Physical assessment
[0177] Echocardiography
[0178] AICD monitoring
[0179] ECG
Efficacy:
[0180] Change from baseline in LVESV, LVEDV, LVEF, and wall motion
score index
[0181] Change from baseline in NYHA classification and quality of
life
[0182] Change from baseline in perfusion as determined by SPECT
imaging
[0183] Change from baseline in Six Minute Walk Test distance
TABLE-US-00003 TABLE 2 Clinical Dosing Schedule # of Amount of
Injection # Injection Total Dose Cohort Subjects DNA/site
volume/site Sites of DNA Cohort A 4 0.33 mg 1.0 ml 15 5 mg Cohort B
6 1.0 mg 1.0 ml 15 15 mg Cohort C 6 2.0 mg 1.0 ml 15 30 mg
[0184] Based on preclinical data, delivery of JVS-100 is expected
to elicit an improvement cardiac function and symptoms at 4 months
that sustains to 12 months. At 4 months following JVS-100
injection, compared to baseline values, an improvement in six
minute walk distance of about greater than 30 meters, an
improvement in quality of life score of about 10%, and/or an
improvement of approximately 1 NYHA class are anticipated.
Similarly, we expect a relative improvement in LVESV, LVEF, and/or
WMSI of approximately 10% compared to baseline values.
Comparative Example 1
[0185] Evaluation of Cardiac Function by Echocardiography in
Chronic Heart Failure Pigs after Treatment with ACL-01110Sk or
ACL-01010Sk
Purpose
[0186] The purpose of this study is to compare functional cardiac
response to SDF-1 plasmids ACL-01110Sk or ACL-01010Sk after
endomyocardial catheter delivery in a porcine model of ischemic
heart failure
[0187] This study compared efficacy of ACL-01110Sk and ACL-01010Sk
in improving function in a porcine ischemic heart failure model. In
this study, the plasmids were delivered by an endoventricular
needle injection catheter. Efficacy was assessed by measuring the
impact of the therapy on cardiac remodeling (i.e., left ventricular
volumes) and function (i.e., left ventricular ejection fraction
(LVEF)) via echocardiography.
Methods
[0188] Briefly, male Yorkshire pigs were given myocardial
infarctions by LAD occlusion via balloon angioplasty for 90
minutes. Pigs having an ejection fraction <40% as measured by
M-mode echocardiography 30 days post-infarct were enrolled. Pigs
were randomized to one of 3 groups to be injected with either
Phosphate Buffered Saline (PBS, control), ACL-01110Sk in PBS, or
ACL-01010Sk in PBS using an endoventricular needle injection
catheter delivery system (Table 3).
TABLE-US-00004 TABLE 3 Initial Study Design: SDF-1 Therapy for
Chronic Heart Failure in Pigs # of Injection Amount of # Injection
Total Group Plasmid Pigs volume/site DNA/site Sites DNA 1 Vehicle 3
200 .mu.l N/A 10 n/a 2 ACL- 3 200 .mu.l 400 .mu.g 10 4 mg 01010Sk 3
ACL- 3 200 .mu.l 400 .mu.g 10 4 mg 01110Sk
[0189] Echocardiograms were recorded prior to injection and at 30
and 60 days post-injection. Table 8 below defines the variables as
they are referred to in this report.
TABLE-US-00005 TABLE 4 Definition of variables Variable Name
Definition LVESV End Systolic Volume measured in parasternal
long-axis view LVEDV End Diastolic Volume measured in parasternal
long-axis view LVEF (LVEDV - LVESV)/LVEDV * 100%
Results
[0190] The baseline echocardiographic characteristics at time of
initial injection (Day 30 post-MI) for all enrolled animals in this
report (n=9) as reported by the echocardiography core laboratory,
are provided in Table 5 below.
TABLE-US-00006 TABLE 5 Baseline characteristics Baseline Value
Baseline Value Parameter Group 1 Group 2 Baseline Value Group 3
LVESV 78 .+-. 18 ml 67 .+-. 2 ml 86 .+-. 31 ml LVEDV 132 .+-. 30 ml
114 .+-. 11 ml 130 .+-. 36 ml LVEF 41 .+-. 1% 41 .+-. 5% 34 .+-.
10%
[0191] Table 5 shows the LVESV, LVEF and LVEDV at 0 and 30 days
post-initial injection. Control PBS animals demonstrated an
increase in LVESV and LVEDV and no improvement in LVEF consistent
with this heart failure model. The treatment groups did not reduce
cardiac volumes or increase LVEF compared to control. Similar
results were obtained at 60 days post-initial injection.
Comparative Example 2
[0192] A strategy to augment stem cell homing to the periinfarct
region by catheter-based transendocardial delivery of SDF-1 in a
porcine model of myocardial infarction was investigated to
determine if it would improve left ventricular perfusion and
function. The catheter-based approach has been used successfully
for cell transplantation and delivery of angiogenic growth factors
in humans.
[0193] Female German landrace pigs (30 kg) were used. After an
overnight fast, animals were anesthetized and intubated.
[0194] A 7 French sheath was placed in the femoral artery with the
animal in a supine position. An over-the-wire balloon was advanced
to the distal LAD. The balloon was inflated with 2 atm and agarose
beads were injected slowly over 1 min via the balloon catheter into
the distal LAD. After 1 minute the balloon was deflated and the
occlusion of the distal LAD was documented by angiography. After
induction of myocardial infarction animals were monitored for 3-4 h
until rhythm and blood pressure was stable. The arterial sheath was
removed, carprofen (4 mg/kg) was administered intramuscularly and
the animals were weaned from the respirator. Two weeks after
myocardial infarction animals were anesthetized. Electromechanical
mapping of the left ventricle was performed via an 8F femoral
sheath with the animal in the supine position. After a complete map
of the left ventricle had been obtained, human SDF-1 (Peprotec,
Rocky-Hill, N.J.) was delivered by 18 injections (5 .mu.g in 100
.mu.ml saline) into the infarct and periinfarct region via an
injection catheter. 5 .mu.g per injection was used to adjust for
the reported efficiency of the catheter injection. Injections were
performed slowly over 20 s and only when the catheter's tip was
perpendicular) to the left ventricular wall, when loop stability
was <2 mm and when needle protrusion into the myocardium
provoked ectopic ventricular extra beats. Control animals underwent
an identical procedure with sham injections. Echocardiography
excluded postinterventional pericardial effusion.
[0195] Twenty (20) animals completed the study protocol: 8 control
animals and 12 SDF-1 treated animals. For myocardial perfusion
imaging only 6 control animals could be evaluated due to technical
problems. Infarct location was anteroseptal in all animals.
[0196] Infarct size in percent of the left ventricle as determined
by tetrazolium staining was 8.9.+-.2.6% in the control group and
8.9.+-.1.2% in the SDF-1 group. Left ventricular muscle volume was
similar in both groups (83.+-.14 ml versus 95.+-.10 ml, p=ns).
Immunofluorescence staining revealed significantly more
vWF-positive vessels in the peri-infarct area in SDF-1 treated
animals than in control animals (349.+-.17/mm.sup.2 vs.
276.+-.21/mm.sup.2, p<0.05). A profound loss of collagen in the
periinfarct area was observed in SDF-1 treated animals as compared
to control animals (32.+-.5% vs. 61.+-.6%, p<0.005). The number
of inflammatory cells (neutrophils and macrophages) within the
periinfarct area was similar in both groups (332.+-.51/mm.sup.2 vs.
303.+-.55/mm.sup.2, p=ns). Global myocardial perfusion did not
change from baseline to follow-up SPECT and there was no difference
between groups. Final infarct size was similar in both groups and
compared well to the results of tetrazolium staining. Segmental
analysis of myocardial perfusion revealed decreased tracer uptake
in apical and anteroseptal segments with significant differences
between myocardial segments. However, tracer uptake at baseline and
follow-up were nearly identical in control and SDF-1 treated
animals. There were no differences in end diastolic and end
systolic volumes between groups. However, stroke volume increased
in control animals and decreased slightly in SDF-1 treated animals.
The difference between both groups was significant.
[0197] Similarly, ejection fraction increased in control animals
and decreased in SDF-1 treated animals. The difference between
groups showed a strong trend (p=0.05). Local shortening, another
parameter of ventricular mechanical function, did not change in
control animals. However, local shortening decreased significantly
in SDF-1 treated animals, resulting in a significant difference
between groups. There were no significant differences in unipolar
voltage within and between groups. Significant correlations between
baseline ejection fraction and stroke volume and baseline local
shortening (EF and LS: r=0.71, SV and LS: r=0.59) were noted.
Similar results were obtained for follow-up values (EF and LS:
r=0.49, SV and LS: r=0.46). The change in local shortening
correlated significantly with the change in ejection fraction
(r=0.52) and stroke volume (r=0.46). There was neither a
correlation between local shortening and enddiastolic volume
(baseline r=-0.03, follow-up r=0.12) nor between ejection fraction
and enddiastolic volume (baseline r=-0.04, follow-up r=0.05).
Segmental analysis of EEM data showed decreased unipolar voltage
and local shortening in the anteroseptal segments with significant
differences between myocardial segments at baseline. The
distribution of unipolar voltage values in myocardial segments was
similar in both groups at baseline and at follow-up. Segmental
local shortening did not change in the control group. However, it
decreased in the SDF-1 group, mainly due to a decrease in the
lateral and posterior segment of the left ventricle. There was a
significant interaction between assignment to SDF-1 and follow-up
vs. baseline.
[0198] The study described above demonstrated that a single
application of SDF-1 protein was insufficient to produce functional
cardiac benefit.
[0199] From the above description of the application, those skilled
in the art will perceive improvements, changes and modifications.
Such improvements, changes and modifications within the skill of
the art are intended to be covered by the appended claims. All
patents, patent applications and publications cited herein are
incorporated by reference in their entirety.
Sequence CWU 1
1
5168PRTArtificialPolypeptide 1Lys Pro Val Ser Leu Leu Tyr Arg Cys
Pro Cys Arg Phe Phe Glu Ser1 5 10 15His Val Ala Arg Ala Asn Val Lys
His Leu Lys Ile Leu Asn Thr Pro 20 25 30Asn Cys Ala Leu Gln Ile Val
Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45Val Cys Ile Asp Pro Lys
Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50 55 60Ala Leu Asn
Lys65289PRTHomo sapiens 2Met Asn Ala Lys Val Val Val Val Leu Val
Leu Val Leu Thr Ala Leu1 5 10 15Cys Leu Ser Asp Gly Lys Pro Val Ser
Leu Ser Tyr Arg Cys Pro Cys 20 25 30Arg Phe Phe Glu Ser His Val Ala
Arg Ala Asn Val Lys His Leu Lys 35 40 45Ile Leu Asn Thr Pro Asn Cys
Ala Leu Gln Ile Val Ala Arg Leu Lys 50 55 60Asn Asn Asn Arg Gln Val
Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln65 70 75 80Glu Tyr Leu Glu
Lys Ala Leu Asn Lys 85389PRTRattus norvegicus 3Met Asp Ala Lys Val
Val Ala Val Leu Ala Leu Val Leu Ala Ala Leu1 5 10 15Cys Ile Ser Asp
Gly Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys 20 25 30Arg Phe Phe
Glu Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys 35 40 45Ile Leu
Asn Thr Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys 50 55 60Ser
Asn Asn Arg Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln65 70 75
80Glu Tyr Leu Asp Lys Ala Leu Asn Lys 8541940DNAHomo sapiens
4gccgcacttt cactctccgt cagccgcatt gcccgctcgg cgtccggccc ccgacccgcg
60ctcgtccgcc cgcccgcccg cccgcccgcg ccatgaacgc caaggtcgtg gtcgtgctgg
120tcctcgtgct gaccgcgctc tgcctcagcg acgggaagcc cgtcagcctg
agctacagat 180gcccatgccg attcttcgaa agccatgttg ccagagccaa
cgtcaagcat ctcaaaattc 240tcaacactcc aaactgtgcc cttcagattg
tagcccggct gaagaacaac aacagacaag 300tgtgcattga cccgaagcta
aagtggattc aggagtacct ggagaaagct ttaaacaagt 360aagcacaaca
gccaaaaagg actttccgct agacccactc gaggaaaact aaaaccttgt
420gagagatgaa agggcaaaga cgtgggggag ggggccttaa ccatgaggac
caggtgtgtg 480tgtggggtgg gcacattgat ctgggatcgg gcctgaggtt
tgccagcatt tagaccctgc 540atttatagca tacggtatga tattgcagct
tatattcatc catgccctgt acctgtgcac 600gttggaactt ttattactgg
ggtttttcta agaaagaaat tgtattatca acagcatttt 660caagcagtta
gttccttcat gatcatcaca atcatcatca ttctcattct cattttttaa
720atcaacgagt acttcaagat ctgaatttgg cttgtttgga gcatctcctc
tgctcccctg 780gggagtctgg gcacagtcag gtggtggctt aacagggagc
tggaaaaagt gtcctttctt 840cagacactga ggctcccgca gcagcgcccc
tcccaagagg aaggcctctg tggcactcag 900ataccgactg gggctgggcg
ccgccactgc cttcacctcc tctttcaacc tcagtgattg 960gctctgtggg
ctccatgtag aagccactat tactgggact gtgctcagag acccctctcc
1020cagctattcc tactctctcc ccgactccga gagcatgctt aatcttgctt
ctgcttctca 1080tttctgtagc ctgatcagcg ccgcaccagc cgggaagagg
gtgattgctg gggctcgtgc 1140cctgcatccc tctcctccca gggcctgccc
cacagctcgg gccctctgtg agatccgtct 1200ttggcctcct ccagaatgga
gctggccctc tcctggggat gtgtaatggt ccccctgctt 1260acccgcaaaa
gacaagtctt tacagaatca aatgcaattt taaatctgag agctcgcttt
1320gagtgactgg gttttgtgat tgcctctgaa gcctatgtat gccatggagg
cactaacaaa 1380ctctgaggtt tccgaaatca gaagcgaaaa aatcagtgaa
taaaccatca tcttgccact 1440accccctcct gaagccacag cagggtttca
ggttccaatc agaactgttg gcaaggtgac 1500atttccatgc ataaatgcga
tccacagaag gtcctggtgg tatttgtaac tttttgcaag 1560gcattttttt
atatatattt ttgtgcacat ttttttttac gtttctttag aaaacaaatg
1620tatttcaaaa tatatttata gtcgaacaat tcatatattt gaagtggagc
catatgaatg 1680tcagtagttt atacttctct attatctcaa actactggca
atttgtaaag aaatatatat 1740gatatataaa tgtgattgca gcttttcaat
gttagccaca gtgtattttt tcacttgtac 1800taaaattgta tcaaatgtga
cattatatgc actagcaata aaatgctaat tgtttcatgg 1860tataaacgtc
ctactgtatg tgggaattta tttacctgaa ataaaattca ttagttgtta
1920gtgatggagc ttaaaaaaaa 19405293DNARattus norvegicus 5ccatggacgc
caaggtcgtc gctgtgctgg ccctggtgct ggccgcgctc tgcatcagtg 60acggtaagcc
agtcagcctg agctacagat gcccctgccg attctttgag agccatgtcg
120ccagagccaa cgtcaaacat ctgaaaatcc tcaacactcc aaactgtgcc
cttcagattg 180ttgcaaggct gaaaagcaac aacagacaag tgtgcattga
cccgaaatta aagtggatcc 240aagagtacct ggacaaagcc ttaaacaagt
aagcacaaca gcccaaagga ctt 293
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