U.S. patent application number 14/378645 was filed with the patent office on 2015-04-30 for systemic delivery and regulated expression of paracrine genes for cardiovascular diseases and other conditions.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Mei Hua Gao, H. Kirk Hammond.
Application Number | 20150118287 14/378645 |
Document ID | / |
Family ID | 48984885 |
Filed Date | 2015-04-30 |
United States Patent
Application |
20150118287 |
Kind Code |
A1 |
Hammond; H. Kirk ; et
al. |
April 30, 2015 |
SYSTEMIC DELIVERY AND REGULATED EXPRESSION OF PARACRINE GENES FOR
CARDIOVASCULAR DISEASES AND OTHER CONDITIONS
Abstract
In alternative embodiments, the invention provides methods for
treating, ameliorating or protecting (preventing) an individual or
a patient against a disease, an infection or a condition responsive
to an increased paracrine polypeptide level in vivo comprising:
providing a paracrine polypeptide-encoding nucleic acid or gene
operatively linked to a transcriptional regulatory sequence; or an
expression vehicle, a vector, a recombinant virus, or equivalent,
having contained therein a paracrine-encoding nucleic acid or gene,
and the expression vehicle, vector, recombinant virus, or
equivalent can express the paracrine-encoding nucleic acid or gene
in a cell or in vivo; and administering or delivering the paracrine
polypeptide-encoding nucleic acid or gene operatively linked to a
transcriptional regulatory sequence, or the expression vehicle,
vector, recombinant virus, or equivalent, to an individual or a
patient in need thereof, thereby treating, ameliorating or
protecting (preventing) the individual or patient against the
disease, infection or condition responsive to an increased
paracrine polypeptide level.
Inventors: |
Hammond; H. Kirk; (La Jolla,
CA) ; Gao; Mei Hua; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA |
Oakland |
CA |
US |
|
|
Family ID: |
48984885 |
Appl. No.: |
14/378645 |
Filed: |
February 13, 2013 |
PCT Filed: |
February 13, 2013 |
PCT NO: |
PCT/US2013/025997 |
371 Date: |
August 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61598772 |
Feb 14, 2012 |
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|
Current U.S.
Class: |
424/450 ;
424/93.2; 424/93.21; 514/4.9; 514/44R; 514/5.2; 514/6.9 |
Current CPC
Class: |
C12N 15/86 20130101;
C12N 2710/10343 20130101; A61P 1/16 20180101; A61K 38/2228
20130101; A61K 31/65 20130101; A61K 38/30 20130101; A61P 9/00
20180101; A61P 9/04 20180101; A61P 31/00 20180101; C12N 2830/003
20130101; A61K 47/6901 20170801; A61P 1/18 20180101; A61P 31/04
20180101; C07K 14/075 20130101; A61P 21/00 20180101; C12N 15/861
20130101; A61P 9/12 20180101; A61P 13/12 20180101; A61P 35/00
20180101; A61K 48/005 20130101; A61P 17/00 20180101; A61K 38/2221
20130101; A61P 43/00 20180101; A61P 11/00 20180101; C12N 2750/14143
20130101; A61K 38/2242 20130101; A61P 3/04 20180101; A61K 31/436
20130101; A61K 38/25 20130101; C07K 14/57509 20130101; A61K 48/00
20130101; A61P 3/10 20180101; A61P 25/00 20180101; C07K 14/65
20130101; A61K 38/52 20130101 |
Class at
Publication: |
424/450 ;
514/44.R; 424/93.2; 424/93.21; 514/6.9; 514/5.2; 514/4.9 |
International
Class: |
A61K 38/22 20060101
A61K038/22; A61K 31/436 20060101 A61K031/436; A61K 31/65 20060101
A61K031/65; A61K 47/48 20060101 A61K047/48 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0001] This invention was made with government support under grant
no. HL088426 awarded by the National Institutes of Health (NIH),
DHHS. The government has certain rights in the invention.
Claims
1. A method for treating, ameliorating, protecting or preventing an
individual or a patient against a disease, an infection or a
condition responsive to an increased or sustained peptide or
paracrine polypeptide level in vivo comprising: (i) providing a
paracrine polypeptide-encoding nucleic acid or gene operatively
linked to a transcriptional regulatory sequence; or an expression
vehicle, a vector, a recombinant virus, or equivalent, having
contained therein a paracrine-encoding nucleic acid or gene, or a
paracrine polypeptide-expressing nucleic acid, transcript or
message, and the expression vehicle, vector, recombinant virus, or
equivalent can express the paracrine-encoding nucleic acid, gene,
transcript or message in a cell or in vivo; and (ii) administering
or delivering the paracrine polypeptide-encoding nucleic acid,
gene, transcript or message operatively linked to a transcriptional
regulatory sequence, or the expression vehicle, vector, recombinant
virus, or equivalent, to the cell, or an individual or a patient in
need thereof, thereby treating, ameliorating, protecting or
preventing the individual or patient against the disease, infection
or condition responsive to an increased or a sustained paracrine
polypeptide level.
2. The method of claim 1, wherein: (a) the paracrine-encoding
nucleic acid or gene operatively linked to the transcriptional
regulatory sequence; or the expression vehicle, vector, recombinant
virus, or equivalent, is administered or delivered to the
individual or a patient in need thereof, by oral, intramuscular
(IM) injection, by intravenous (IV) injection, by subcutaneous (SC)
or intradermal injection, by intrathecal injection, by
intra-arterial (IA) injection, by intracoronary injection, by
inhalation, by aerosol, or by a biolistic particle delivery system,
or by using a "gene gun", air pistol or a HELIOS.TM. gene gun
(Bio-Rad Laboratories, Hercules, Calif.); or (b) the
paracrine-encoding nucleic acid or gene operatively linked to the
transcriptional regulatory sequence; or the expression vehicle,
vector, recombinant virus, or equivalent, is administered or
delivered to the individual or a patient in need thereof, by
introduction into any tissue or fluid space within the body that is
adjacent to or is drained by the bloodstream, such that the encoded
protein may be secreted from cells in the tissue and released into
the bloodstream.
3. The method of claim 1, wherein the paracrine polypeptide or
peptide is or comprises: a mammalian cardiotonic peptide, a growth
factor, a Serelaxin, a Relaxin-2, a Urocortin-2 (UCn-2), a
Urocortin-1 (UCn-1), a Urocortin-3 (UCn-3), a Brain Natriuretic
Peptide, a Prostacyclin Synthase, a Growth Hormone, an Insulin-like
Growth Factor-1, or any combination thereof; or, a human
cardiotonic peptide, a human growth factor, a Serelaxin, a
Relaxin-2, a Urocortin-2, a Urocortin-1, a Urocortin-3, a Brain
Natriuretic Peptide, a Prostacyclin Synthase, a Growth Hormone, an
Insulin-like Growth Factor-11, or any combination thereof.
4. The method of claim 3, wherein the paracrine polypeptide is a
Urocortin, a Urocortin-2, a Urocortin-1, a Urocortin-3, a Relaxin-2
or a Brain Natriuretic Peptide and the disease or condition is a
congestive heart failure (CHF); or the paracrine polypeptide is
Prostacyclin Synthase and the disease or condition a pulmonary
hypertension.
5. The method of claim 1, wherein: (a) the individual, patient or
subject is administered a stimulus or signal that induces
expression of the paracrine-expressing nucleic acid or gene, or
induces or activates a promoter operably linked to the
paracrine-expressing nucleic acid or gene that induces expression
of the paracrine-expressing nucleic acid or gene; (b) the
individual, patient or subject is administered a stimulus or signal
that induces synthesis of an activator of a promoter, optionally a
paracrine-expressing nucleic acid or gene-specific promoter
operably linked to the paracrine-expressing nucleic acid or gene;
(c) the individual, patient or subject is administered a stimulus
or signal that induces synthesis of a natural or a synthetic
activator of the paracrine-expressing nucleic acid or gene or the
paracrine-expressing nucleic acid or gene-specific promoter,
wherein optionally the natural activator is an endogenous
transcription factor; (d) the method of (c), wherein the synthetic
activator is a zinc-finger DNA binding protein designed to
specifically and selectively turn on an endogenous or exogenous
target gene, wherein optionally the endogenous target is a gene
paracrine-expressing nucleic acid or gene or an activator of a
paracrine-expressing nucleic acid or gene, or an activator of a
promoter operatively linked to a paracrine-expressing nucleic acid
or gene; (e) the method of any of (a) to (c), wherein the stimulus
or signal comprises a biologic, a light, a chemical or a
pharmaceutical stimulus or signal; (f) the individual, patient or
subject is administered a stimulus or signal that stimulates or
induces expression of a post-transcriptional activator of a
paracrine-expressing nucleic acid or gene, or an activator of a
promoter operatively linked to a paracrine-expressing nucleic acid
or gene, or (g) the individual, patient or subject is administered
a stimulus or signal that inhibits or induces inhibition of a
transcriptional repressor or a post-transcriptional repressor of a
paracrine-expressing nucleic acid or gene.
6. The method of claim 5, wherein the chemical or pharmaceutical
that induces expression of the paracrine-expressing nucleic acid or
gene, or induces expression of the regulated or inducible promoter
operatively linked to the paracrine-expressing nucleic acid or
gene, is an oral antibiotic, a doxycycline or a rapamycin; or a
tet-regulation system using doxycycline is used to induce
expression of the paracrine-expressing nucleic acid or gene, or an
equivalent thereof.
7. The method of claim 1, wherein the paracrine-expressing nucleic
acid or gene or the expression vehicle, vector, recombinant virus,
or equivalent, is formulated in a liquid, a gel, a hydrogel, a
powder or an aqueous or a saline formulation.
8. The method of claim 1, wherein the paracrine-expressing nucleic
acid or gene or the expression vehicle, vector, recombinant virus,
or equivalent, is formulated in a vesicle, liposome, nanoparticle
or nanolipid particle (NLP).
9. The method of claim 1, wherein the paracrine-expressing nucleic
acid or gene or the expression vehicle, vector, recombinant virus,
or equivalent, is formulated in an isolated or cultured cell, and
optionally the cell is a mammalian cell, a cardiac cell, or a human
cell, a non-human primate cell, a monkey cell, a mouse cell, a rat
cell, a guinea pig cell, a rabbit cell, a hamster cell, a goat
cell, a bovine cell, an equine cell, an ovine cell, a canine cell
or a feline cell.
10. The method of claim 1, wherein the paracrine-expressing nucleic
acid or gene or the expression vehicle, vector, recombinant virus,
or equivalent, is formulated as a pharmaceutical or sterile.
11. The method of claim 1, wherein the paracrine-expressing nucleic
acid or gene or the expression vehicle, vector, recombinant virus,
or equivalent, is formulated or delivered with, on, or in
conjunction with a product of manufacture, an artificial organ or
an implant.
12. The method of claim 1, wherein the paracrine-expressing nucleic
acid or gene or the expression vehicle, vector, recombinant virus,
or equivalent expresses a paracrine polypeptide in vitro or ex
vivo.
13. A method for treating, ameliorating, protecting or preventing
an individual or a patient against a paracrine-responsive
pathology, infection, disease, illness, or condition, comprising
practicing the method of claim 1.
14. A method for treating, ameliorating, protecting or preventing a
cardiac contractile dysfunction; a congestive heart failure (CHF);
a cardiac fibrosis; a cardiac myocyte disease, dysfunction or
apoptosis; a pulmonary hypertension; a heart, skin, liver, lung,
muscle, nerve, brain or kidney disease, cancer or dysfunction; a
cancer or a neoplasia; or, a hemophilia or a Hemophilia B,
comprising practicing the method of claim 1.
15. A treating, ameliorating, protecting or preventing a diabetes
or a pre-diabetes in a patient or an individual comprising: (a)
practicing the method of claim 1, wherein the paracrine polypeptide
or peptide comprises or consists of a urocortin-2 (UCn-2); and (b)
administering a urocortin-2 (UCn-2) peptide or polypeptide, or a
nucleic acid, gene, message or transcript encoding a urocortin-2
(UCn-2) to an individual or patient in need thereof, wherein
optionally the urocortin-2 (UCn-2) peptide or polypeptide is an
isolated, a recombinant, a synthetic and/or a peptidomimetic
peptide or polypeptide or variant thereof, thereby treating,
ameliorating, protecting or preventing a the diabetes or
pre-diabetes in the patient or individual.
16. A method of treating, ameliorating, protecting or preventing an
obesity in a patient or an individual comprising: (a) practicing
the method of claim 1, wherein the paracrine polypeptide or peptide
comprises or consists of a urocortin-2 (UCn-2); and (b)
administering a urocortin-2 (UCn-2) peptide or polypeptide, or a
nucleic acid, gene, message or transcript encoding a urocortin-2
(UCn-2) to an individual or patient in need thereof, wherein
optionally the urocortin-2 (UCn-2) peptide or polypeptide is an
isolated, a recombinant, a synthetic and/or a peptidomimetic
peptide or polypeptide or variant thereof, thereby treating,
ameliorating, protecting or preventing a the obesity in the patient
or individual.
17. A method of suppressing weight gain, or suppressing the
appetite, or stimulating or initiating weight loss, in a patient or
an individual comprising: (a) practicing the method of claim 1,
wherein the paracrine polypeptide or peptide comprises or consists
of a urocortin-2 (UCn-2); and (b) administering a urocortin-2
(UCn-2) peptide or polypeptide, or a nucleic acid, gene, message or
transcript encoding a urocortin-2 (UCn-2) to an individual or
patient in need thereof, wherein optionally the urocortin-2 (UCn-2)
peptide or polypeptide is an isolated, a recombinant, a synthetic
and/or a peptidomimetic peptide or polypeptide or variant thereof,
thereby suppressing weight gain, or suppressing the appetite, or
stimulating or initiating weight loss, in the patient or
individual.
18. The method of claim 10, wherein the urocortin-2 (UCn-2) peptide
or polypeptide is formulated in or as a vesicle, liposome,
nanoparticle or nanolipid particle (NLP), or is formulated for:
oral administration, intramuscular (IM) injection, intravenous (IV)
injection, subcutaneous (SC) or intradermal injection, intrathecal
injection, intra-arterial (IA) injection, intracoronary injection,
inhalation, or administration by aerosol.
19. The method of claim 1, wherein the expression vehicle, vector,
recombinant virus, or equivalent is or comprises: an
adeno-associated virus (AAV), a lentiviral vector or an adenovirus
vector, an AAV serotype AAV5, AAV6, AAV8 or AAV9, a rhesus-derived
AAV, or the rhesus-derived AAV AAVrh.10hCLN2, an AAV capsid mutant
or AAV hybrid serotype, an organ-tropic AAV, or a cardiotropic AAV,
or a cardiotropic AAVM41 mutant, wherein optionally the AAV is
engineered to increase efficiency in targeting a specific cell type
that is non-permissive to a wild type (wt) AAV and/or to improve
efficacy in infecting only a cell type of interest, and optionally
the hybrid AAV is retargeted or engineered as a hybrid serotype by
one or more modifications comprising: 1) a transcapsidation, 2)
adsorption of a bi-specific antibody to a capsid surface, 3)
engineering a mosaic capsid, and/or 4) engineering a chimeric
capsid.
20. The method of claim 1, wherein: (a) the paracrine-encoding
nucleic acid, gene, transcript or message is operatively linked to
a regulated or inducible transcriptional regulatory sequence; (b)
the regulated or inducible transcriptional regulatory sequence is a
regulated or inducible promoter, wherein optionally a positive or
an activator and/or a negative or a repressor modulator of
transcription and/or translation is operably linked to the
paracrine polypeptide-encoding nucleic acid, gene, transcript or
message; (c) administering the paracrine polypeptide-encoding
nucleic acid, gene, transcript or message operatively linked to a
transcriptional regulatory sequence, or the expression vehicle,
vector, recombinant virus, or equivalent, to an individual or a
patient in need thereof results in a paracrine protein being
released into the bloodstream or general circulation, or an
increased or sustained expression of the paracrine protein in the
cell, wherein optionally the release or increased or sustained
expression of the paracrine protein is dependent on activation of
an inducible promoter, or de-repression of a repressor, operably
linked to the paracrine polypeptide-encoding nucleic acid, gene,
transcript or message; or (d) the method of any of (a) to (c),
wherein the disease, infection or condition responsive to an
increased paracrine polypeptide level in vivo is a cardiac
contractile dysfunction; a congestive heart failure (CHF); a
cardiac fibrosis; a cardiac myocyte disease, dysfunction or
apoptosis; a pulmonary hypertension; a heart, skin, liver, lung,
muscle, nerve, brain or kidney disease, cancer or dysfunction; a
cancer or a neoplasia; or, a hemophilia or a Hemophilia B.
Description
TECHNICAL FIELD
[0002] This invention relates to cellular and molecular biology and
medicine. The invention provides compositions and in vitro and ex
vivo methods. In alternative embodiments, the invention provides
methods for treating, ameliorating or protecting (preventing) an
individual or a patient against a disease, an infection or a
condition responsive to an increased or a sustained paracrine
polypeptide level in vivo comprising: providing a paracrine
polypeptide-encoding nucleic acid or gene operatively linked to a
transcriptional regulatory sequence; or an expression vehicle, a
vector, a recombinant virus, or equivalent, having contained
therein a paracrine-encoding nucleic acid, gene, transcript or
message, and the expression vehicle, vector, recombinant virus, or
equivalent can express the paracrine-encoding encoding nucleic
acid, gene, transcript or message in a cell or in vivo; and
administering or delivering the paracrine polypeptide-encoding
nucleic acid, gene, transcript or message operatively linked to a
transcriptional regulatory sequence, or the expression vehicle,
vector, recombinant virus, or equivalent, to an individual or a
patient in need thereof, thereby treating, ameliorating or
protecting (preventing) the individual or patient against the
disease, infection or condition responsive to an increased
paracrine polypeptide level.
BACKGROUND
[0003] Recently an intravenous injection of a virus vector encoding
human Factor IX, which is deficient in Hemophilia B was shown to
increase Factor IX concentration in the serum of subjects with
Hemophilia B to a degree that lowered their requirements for
exogenous Factor IX infusion. However: 1) this protein was not
under regulated expression, and therefore, did not enable optimal
tailoring of levels of the transgene in the serum, 2) this system
did not provide for a means to turn off transgene expression in
case of undesired or unexpected effects, and 3) the gene, Factor
IX, was not a paracrine gene, and had no beneficial cardiovascular
effects, and therefore, could not be used to treat heart
disease.
SUMMARY
[0004] The invention provides methods for treating, ameliorating or
protecting (preventing) an individual or a patient against any
disease, infection or condition responsive to an increased
paracrine polypeptide level in vivo. In alternative embodiments,
the invention provides methods for treating, ameliorating or
protecting (preventing) against a disease, an infection or a
condition responsive to an increased or sustained peptide or
paracrine polypeptide level in vivo comprising:
[0005] (a) (i) providing a paracrine polypeptide-encoding nucleic
acid or gene operatively linked to a transcriptional regulatory
sequence; or an expression vehicle, a vector, a recombinant virus,
or equivalent, having contained therein a paracrine-encoding
nucleic acid or gene, or a paracrine polypeptide-expressing nucleic
acid, transcript or message, and the expression vehicle, vector,
recombinant virus, or equivalent can express the paracrine-encoding
nucleic acid, gene, transcript or message in a cell or in vivo;
and
[0006] (ii) administering or delivering the paracrine
polypeptide-encoding nucleic acid, gene, transcript or message
operatively linked to a transcriptional regulatory sequence, or the
expression vehicle, vector, recombinant virus, or equivalent, to
the cell, or an individual or a patient in need thereof,
[0007] thereby treating, ameliorating or protecting (preventing)
the individual or patient against the disease, infection or
condition responsive to an increased or a sustained paracrine
polypeptide level;
[0008] (b) the method of (a), wherein the expression vehicle,
vector, recombinant virus, or equivalent is or comprises:
[0009] an adeno-associated virus (AAV), a lentiviral vector or an
adenovirus vector,
[0010] an AAV serotype AAV5, AAV6, AAV8 or AAV9,
[0011] a rhesus-derived AAV, or the rhesus-derived AAV
AAVrh.10hCLN2,
[0012] an AAV capsid mutant or AAV hybrid serotype,
[0013] an organ-tropic AAV, or a cardiotropic AAV, or a
cardiotropic AAVM41 mutant,
[0014] wherein optionally the AAV is engineered to increase
efficiency in targeting a specific cell type that is non-permissive
to a wild type (wt) AAV and/or to improve efficacy in infecting
only a cell type of interest,
[0015] and optionally the hybrid AAV is retargeted or engineered as
a hybrid serotype by one or more modifications comprising: 1) a
transcapsidation, 2) adsorption of a bi-specific antibody to a
capsid surface, 3) engineering a mosaic capsid, and/or 4)
engineering a chimeric capsid;
[0016] (c) the method of (a), wherein the paracrine-encoding
nucleic acid, gene, transcript or message is operatively linked to
a regulated or inducible transcriptional regulatory sequence;
[0017] (d) the method of (c), wherein the regulated or inducible
transcriptional regulatory sequence is a regulated or inducible
promoter,
[0018] wherein optionally a positive (an activator) and/or a
negative (a repressor) modulator of transcription and/or
translation is operably linked to the paracrine
polypeptide-encoding nucleic acid, gene, transcript or message;
[0019] (e) the method of any of (a) to (d), wherein administering
the paracrine polypeptide-encoding nucleic acid, gene, transcript
or message operatively linked to a transcriptional regulatory
sequence, or the expression vehicle, vector, recombinant virus, or
equivalent, to an individual or a patient in need thereof results
in a paracrine protein being released into the bloodstream or
general circulation, or an increased or sustained expression of the
paracrine protein in the cell,
[0020] wherein optionally the release or increased or sustained
expression of the paracrine protein is dependent on activation of
an inducible promoter, or de-repression of a repressor, operably
linked to the paracrine polypeptide-encoding nucleic acid, gene,
transcript or message; or
[0021] (f) the method of any of (a) to (e), wherein the disease,
infection or condition responsive to an increased paracrine
polypeptide level in vivo is a cardiac contractile dysfunction; a
congestive heart failure (CHF); a cardiac fibrosis; a cardiac
myocyte disease, dysfunction or apoptosis; a pulmonary
hypertension; a heart, skin, liver, lung, muscle, nerve, brain or
kidney disease, cancer or dysfunction; a cancer or a neoplasia; or,
a hemophilia or a Hemophilia B.
[0022] In alternative embodiments of methods of the invention:
[0023] (a) the paracrine-encoding nucleic acid or gene operatively
linked to the transcriptional regulatory sequence; or the
expression vehicle, vector, recombinant virus, or equivalent, is
administered or delivered to the individual or a patient in need
thereof, by oral, intramuscular (IM) injection, by intravenous (IV)
injection, by subcutaneous (SC) or intradermal injection, by
intrathecal injection, by intra-arterial (IA) injection, by
intracoronary injection, by inhalation, or by a biolistic particle
delivery system, or by using a "gene gun", air pistol or a
HELIOS.TM. gene gun (Bio-Rad Laboratories, Hercules, Calif.);
or
[0024] (b) the paracrine-encoding nucleic acid or gene operatively
linked to the transcriptional regulatory sequence; or the
expression vehicle, vector, recombinant virus, or equivalent, is
administered or delivered to the individual or a patient in need
thereof, by introduction into any tissue or fluid space within the
body that is adjacent to or is drained by the bloodstream, such
that the encoded protein may be secreted from cells in the tissue
and released into the bloodstream.
[0025] In alternative embodiments of methods of the invention: the
paracrine polypeptide or peptide is or comprises: a mammalian
cardiotonic peptide, a growth factor, a Serelaxin, a Relaxin-2, a
Urocortin-2 (UCn-2), a Urocortin-1 (UCn-1), a Urocortin-3 (UCn-3),
a Brain Natriuretic Peptide, a Prostacyclin Synthase, a Growth
Hormone, an Insulin-like Growth Factor-1, or any combination
thereof; or, a human cardiotonic peptide, a human growth factor, a
Serelaxin, a Relaxin-2, a Urocortin-2, a Urocortin-1, a
Urocortin-3, a Brain Natriuretic Peptide, a Prostacyclin Synthase,
a Growth Hormone, an Insulin-like Growth Factor-11, or any
combination thereof.
[0026] In alternative embodiments of methods of the invention: the
paracrine polypeptide is a Urocortin, a Urocortin-2, a Urocortin-1,
a Urocortin-3, a Relaxin-2 or a Brain Natriuretic Peptide and the
disease or condition is a congestive heart failure (CHF); or the
paracrine polypeptide is Prostacyclin Synthase and the disease or
condition a pulmonary hypertension and the disease or condition is
a congestive heart failure (CHF); or the paracrine polypeptide is
Prostacyclin Synthase and the disease or condition a pulmonary
hypertension.
[0027] In alternative embodiments of methods of the invention:
[0028] (a) the individual, patient or subject is administered a
stimulus or signal that induces expression of the
paracrine-expressing nucleic acid or gene, or induces or activates
a promoter (e.g., operably linked to the paracrine-expressing
nucleic acid or gene) that induces expression of the
paracrine-expressing nucleic acid or gene;
[0029] (b) the individual, patient or subject is administered a
stimulus or signal that induces synthesis of an activator of a
promoter, optionally a paracrine-expressing nucleic acid or
gene-specific promoter (e.g., operably linked to the
paracrine-expressing nucleic acid or gene);
[0030] (c) the individual, patient or subject is administered a
stimulus or signal that induces synthesis of a natural or a
synthetic activator of the paracrine-expressing nucleic acid or
gene or the paracrine-expressing nucleic acid or gene-specific
promoter,
[0031] wherein optionally the natural activator is an endogenous
transcription factor;
[0032] (d) the method of (c), wherein the synthetic activator is a
zinc-finger DNA binding protein designed to specifically and
selectively turn on an endogenous or exogenous target gene, wherein
optionally the endogenous target is a gene paracrine-expressing
nucleic acid or gene or an activator of a paracrine-expressing
nucleic acid or gene, or an activator of a promoter operatively
linked to a paracrine-expressing nucleic acid or gene;
[0033] (e) the method of any of (a) to (c), wherein the stimulus or
signal comprises a biologic, a light, a chemical or a
pharmaceutical stimulus or signal;
[0034] (f) the individual, patient or subject is administered a
stimulus or signal that stimulates or induces expression of a
post-transcriptional activator of a paracrine-expressing nucleic
acid or gene, or an activator of a promoter operatively linked to a
paracrine-expressing nucleic acid or gene, or
[0035] (g) the individual, patient or subject is administered a
stimulus or signal that inhibits or induces inhibition of a
transcriptional repressor or a post-transcriptional repressor of a
paracrine-expressing nucleic acid or gene.
[0036] In alternative embodiments of methods of the invention: the
chemical or pharmaceutical that induces expression of the
paracrine-expressing nucleic acid or gene, or induces expression of
the regulated or inducible promoter operatively linked to the
paracrine-expressing nucleic acid or gene, is an oral antibiotic, a
doxycycline or a rapamycin; or a tet-regulation system using
doxycycline is used to induce expression of the
paracrine-expressing nucleic acid or gene, or an equivalent
thereof.
[0037] In alternative embodiments of methods of the invention: the
paracrine-expressing nucleic acid or gene or the expression
vehicle, vector, recombinant virus, or equivalent, is formulated in
a liquid, a gel, a hydrogel, a powder or an aqueous
formulation.
[0038] In alternative embodiments of methods of the invention: the
paracrine-expressing nucleic acid or gene or the expression
vehicle, vector, recombinant virus, or equivalent, or the
urocortin-2 (UCn-2) peptide or polypeptide, is formulated in a
vesicle, liposome, nanoparticle or nanolipid particle (NLP) or
equivalents, or formulated for delivery using a vesicle, liposome,
nanoparticle or nanolipid particle (NLP) or equivalents.
[0039] In alternative embodiments of methods of the invention: the
paracrine-expressing nucleic acid or gene or the expression
vehicle, vector, recombinant virus, or equivalent, is formulated
in, or inserted or transfected into, an isolated or cultured cell,
and optionally the cell is a mammalian cell, a cardiac cell, or a
human cell, a non-human primate cell, a monkey cell, a mouse cell,
a rat cell, a guinea pig cell, a rabbit cell, a hamster cell, a
goat cell, a bovine cell, an equine cell, an ovine cell, a canine
cell or a feline cell.
[0040] In alternative embodiments of methods of the invention: the
paracrine-expressing nucleic acid or gene or the expression
vehicle, vector, recombinant virus, or equivalent, or the
urocortin-2 (UCn-2) peptide or polypeptide, is formulated as a
pharmaceutical or a sterile formulation.
[0041] In alternative embodiments of methods of the invention: the
paracrine-expressing nucleic acid or gene or the expression
vehicle, vector, recombinant virus, or equivalent, or the
urocortin-2 (UCn-2) peptide or polypeptide, is formulated or
delivered with, on, or in conjunction with a product of
manufacture, an artificial organ or an implant.
[0042] In alternative embodiments of methods of the invention: the
paracrine-expressing nucleic acid or gene or the expression
vehicle, vector, recombinant virus, or equivalent expresses a
paracrine polypeptide in vitro or ex vivo.
[0043] In alternative embodiments the invention provides methods
for treating, ameliorating or protecting (preventing) an individual
or a patient against a paracrine-responsive pathology, infection,
disease, illness, or condition, comprising practicing a method of
the invention.
[0044] In alternative embodiments the invention provides methods
for treating, ameliorating or protecting (preventing) a cardiac
contractile dysfunction; a congestive heart failure (CHF); a
cardiac fibrosis; a cardiac myocyte disease, dysfunction or
apoptosis; a pulmonary hypertension; a heart, skin, liver, lung,
muscle, nerve, brain or kidney disease, cancer or dysfunction; a
cancer or a neoplasia; or, a hemophilia or a Hemophilia B,
comprising practicing a method of the invention.
[0045] In alternative embodiments, the invention provides methods
of treating, ameliorating or protecting (preventing) diabetes or
pre-diabetes in a patient or an individual comprising:
[0046] (a) practicing a method of the invention, wherein the
paracrine polypeptide or peptide comprises or consists of a
urocortin-2 (UCn-2); and
[0047] (b) administering a urocortin-2 (UCn-2) peptide or
polypeptide, or a nucleic acid, gene, message or transcript
encoding a urocortin-2 (UCn-2) to an individual or patient in need
thereof,
[0048] wherein optionally the urocortin-2 (UCn-2) peptide or
polypeptide is an isolated, a recombinant, a synthetic and/or a
peptidomimetic peptide or polypeptide or variant thereof,
[0049] thereby treating, ameliorating or protecting (preventing)
the diabetes or pre-diabetes in the patient or individual.
[0050] In alternative embodiments, the invention provides methods
of treating, ameliorating or protecting (preventing) obesity in a
patient or an individual comprising:
[0051] (a) practicing a method of the invention, wherein the
paracrine polypeptide or peptide comprises or consists of a
urocortin-2 (UCn-2); and
[0052] (b) administering a urocortin-2 (UCn-2) peptide or
polypeptide, or a nucleic acid, gene, message or transcript
encoding a urocortin-2 (UCn-2) to an individual or patient in need
thereof,
[0053] wherein optionally the urocortin-2 (UCn-2) peptide or
polypeptide is an isolated, a recombinant, a synthetic and/or a
peptidomimetic peptide or polypeptide or variant thereof,
[0054] thereby treating, ameliorating or protecting (preventing)
the obesity in the patient or individual.
[0055] In alternative embodiments, the invention provides methods
of suppressing weight gain, or suppressing the appetite, or
stimulating or initiating weight loss, in a patient or an
individual comprising:
[0056] (a) practicing a method of the invention, wherein the
paracrine polypeptide or peptide comprises or consists of a
urocortin-2 (UCn-2); and
[0057] (b) administering a urocortin-2 (UCn-2) peptide or
polypeptide, or a nucleic acid, gene, message or transcript
encoding a urocortin-2 (UCn-2) to an individual or patient in need
thereof,
[0058] wherein optionally the urocortin-2 (UCn-2) peptide or
polypeptide is an isolated, a recombinant, a synthetic and/or a
peptidomimetic peptide or polypeptide or variant thereof,
[0059] thereby suppressing weight gain, or suppressing the
appetite, or stimulating or initiating weight loss, in the patient
or individual.
[0060] In alternative embodiments, the urocortin-2 (UCn-2) peptide
or polypeptide is formulated in or as a vesicle, liposome,
nanoparticle or nanolipid particle (NLP), or is formulated for:
oral administration, intramuscular (IM) injection, intravenous (IV)
injection, subcutaneous (SC) or intradermal injection, intrathecal
injection, intra-arterial (IA) injection, intracoronary injection,
inhalation, or administration by aerosol.
[0061] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
[0062] All publications, patents, patent applications cited herein
are hereby expressly incorporated by reference for all
purposes.
DESCRIPTION OF DRAWINGS
[0063] FIG. 1 illustrates an exemplary construct of the invention
comprising AAV5 encoding IGF 1, as described in Example 2,
below.
[0064] FIG. 2A and FIG. 2B illustrate data from studies where
cultured neonatal rat cardiac myocytes were infected with the
exemplary AAV5.IGFI.tet construct of the invention, and IGFI was
induced, expressed, and then measured, as described in Example 2,
below.
[0065] FIG. 3 graphically illustrates regulated expression of IGFI
mRNA expression in cultured neonatal rat cardiac myocytes after
gene transfer with the exemplary AAV5.IGFI-tet adding, and them
removing, doxicillin, as described in Example 2, below.
[0066] FIG. 4A illustrates photomicrographs showing EGFP expression
in unilateral tibialis anterior muscle 3 weeks after AAV5.EGFP gene
transfer in rats; and FIG. 4B is Table 4, which summarizes data
from the echocardiography measuring the effects of Skeletal Muscle
IGFI Expression in CHF, as described in Example 2, below.
[0067] FIG. 5 illustrates the experimental protocol for gene
transfer of the exemplary AAV5.IGFI.tet of the invention in
skeletal muscle in CHF, as described in Example 2, below.
[0068] FIG. 6 illustrates the effects of AAV5.IGFI-tet gene
transfer on cardiac apoptosis and fibrosis: FIG. 6A graphically
illustrates data from TUNEL staining that indicated that activation
of IGFI expression (IGF-On) was associated with reduced cardiac
myocyte apoptosis; FIG. 6B illustrates picrosirius red-stained
sections of the uninfarcted intraventricular septum from IGF-Off
and IGF-On rats that showed reduced cardiac fibrosis, and collagen
fractional area was reduced; and FIG. 6C graphically illustrates
this data from the IGF-Off and IGF-On rats, as described in Example
2, below.
[0069] FIG. 7 graphically illustrates that intravenous gave better
results than intramuscular administration in increasing serum
levels of IGFI when an exemplary when AAV5 construct of the
invention was administered: intravenous delivery in mice,
intramuscular delivery in rats, as described in Example 2,
below.
[0070] FIG. 8 graphically, and by image, illustrates data showing
the relative efficacy of intravenous delivery of exemplary AAV5 and
AAV9 constructs of the invention using copy number and transgene
expression in liver and heart as endpoints, as described in Example
2, below.
[0071] FIG. 9 illustrates an exemplary protocol for determining and
testing the most appropriate vector to use for a desired or a
particular indication when practicing a method of the invention, as
discussed in Example 2, below.
[0072] FIGS. 10A, 10B, 10C, 10D, 10E, and 10F illustrate exemplary
vector constructs of the invention, as described in Example 2,
below.
[0073] FIG. 11 graphically illustrates data showing that IV AAV8 is
the optimal vector and delivery route to attain sustained increased
levels of serum urocortin-2 (UCn-2) for a paracrine approach, as
described in Example 3, below.
[0074] FIG. 12A graphically illustrates a time course of UCn2 mRNA
expression in liver after IV administration of the exemplary
AAV8.CBA.UCn2 construct; and FIG. 12B graphically illustrates data
showing UCn2 mRNA expression in LV 6 weeks after AAV8.CBA.UCn2 IV
administration, as described in Example 3, below.
[0075] FIG. 13 graphically illustrates data from a study to
determine if UCn2 gene transfer increased LV function by delivery
of the exemplary AAV8.UCn2 construct of the invention by
intravenous (IV) delivery in normal mice: FIG. 13A graphically
illustrates data showing UCn2 gene transfer increased LV
contractile function; FIG. 13B graphically illustrates data showing
-dP/dt also was reduced, indicating enhanced LV relaxation, as
described in Example 3, below.
[0076] FIG. 14 illustrates data showing the effects of UCn2
transfer on the failing heart: FIG. 14A illustrates the study
protocol; and FIGS. 14B and 14C illustrate data showing the effects
of UCn2 transfer on the failing heart, as described in Example 3,
below.
[0077] FIG. 15 illustrates data, FIG. 15A by graph, FIG. 15B by
immunoblot, where normal mice received IV delivery of the exemplary
AAV8.CBA.UCn2; and four weeks later, LV samples from the UCn2 gene
transfer group showed a 2-fold increase in SERCA2a protein
expression, as described in Example 3, below.
[0078] FIG. 16 shows data of Ca.sup.2+ transients following UCn2
gene transfer: FIG. 16A graphically illustrates that UCn2 gene
transfer increased the rate of Ca.sup.2+ decline; FIG. 16B
graphically illustrates that time-to-Ca.sup.2+ transient decay was
shortened in cardiac myocytes from mice that had received UCN2 gene
transfer 4 w prior, as described in Example 3, below.
[0079] FIG. 17 shows data that UCn2 protects cultured neonatal rat
cardiac myocytes from hypoxic injury: FIG. 17A illustrates that
UCn2 preserves morphological normality 24 hr after NaN.sub.3
treatment; FIG. 17B graphically illustrates that UCn2 reduced LDH
release after NaN.sub.3 treatment, as described in Example 3,
below.
[0080] FIG. 18 graphically illustrates that phosphorylation of both
CREB (FIG. 18A) and .beta.-catenin (FIG. 18B) was detected in LV
samples 4 w after IV delivery of the exemplary UCn2.CBA.UCn2
construct of the invention, as described in Example 3, below.
[0081] FIG. 19 illustrates data showing UCn2 affects glucose
regulation: Mice received IV delivery of the exemplary
AAV8.CBA.UCn2: FIG. 19A illustrates that a small reduction in
fasting blood glucose was seen in the UCn2 group: FIG. 19B
illustrates results indicating that UCn2 gene transfer promotes
glucose utilization and protects against diet-induced
hyperglycemia, as described in Example 3, below.
[0082] FIGS. 20A, 20B, 20C, 20D, 20E, and 20F illustrate exemplary
constructs of the invention, as described in Example 3, below.
[0083] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0084] The invention provides compositions and in vivo and ex vivo
methods comprising administration of paracrine-encoding nucleic
acids, genes, transcripts or messages to treat, ameliorate or
protect (as a prophylaxis) individuals against diseases, infections
or conditions responsive to increased paracrine levels in vivo. In
alternative embodiments, the invention provides compositions and
methods for the in vivo or in situ delivery and/or in vivo
expression of, and controlled expression of, any paracrine
polypeptide or peptide, e.g., a mammalian cardiotonic peptide, a
Serelaxin, a Relaxin-2, a Urocortin-2, a Urocortin-1, a
Urocortin-3, a Brain Natriuretic Peptide, a Prostacyclin Synthase,
a Growth Hormone, an Insulin-like Growth Factor-1, or any
combination thereof; or, a human cardiotonic peptide, a Serelaxin,
a Relaxin-2, a Urocortin-2, a Urocortin-1, a Urocortin-3, a Brain
Natriuretic Peptide, a Prostacyclin Synthase, a Growth Hormone, an
Insulin-like Growth Factor-1, or any combination thereof.
[0085] In alternative embodiments, the invention provides
compositions and methods for the delivery and controlled expression
of a paracrine-encoding nucleic acid or gene, or an expression
vehicle (e.g., vector, recombinant virus, and the like) comprising
(having contained therein) a paracrine-encoding nucleic acid or
gene, that results in a paracrine protein being released into the
bloodstream or general circulation where it can have a beneficial
effect on in the body, e.g., such as the heart in the case of
treating cardiovascular disease, or the lungs or kidneys, or other
targets.
[0086] In alternative embodiments, the invention provides
expression vehicles, vectors, recombinant viruses and the like for
in vivo expression of a paracrine-encoding nucleic acid or gene to
practice the methods of this invention. In alternative embodiments,
the expression vehicles, vectors, recombinant viruses and the like
expressing the paracrine-encoding nucleic acid or gene can be
delivered by intramuscular (IM) injection, by intravenous (IV)
injection, by subcutaneous injection, by inhalation, by a biolistic
particle delivery system (e.g., a so-called "gene gun"), and the
like, e.g., as an outpatient, e.g., during an office visit.
[0087] In alternative embodiments, this "peripheral" mode of
delivery, e.g., expression vehicles, vectors, recombinant viruses
and the like injected IM or IV, can circumvent problems encountered
when genes or nucleic acids are expressed directly in an organ
(e.g., the heart, lung or kidney) itself Sustained secretion of a
desired paracrine protein(s) in the bloodstream or general
circulation also circumvents the difficulties and expense of
administering proteins by infusion, which can be particularly
problematic for many proteins which exhibit very short half lives
in the body, as summarized in Table 1, below:
TABLE-US-00001 TABLE 1 Peptide IV Infusion vs Gene Transfer Feature
IV Infusion Gene Transfer Requires Hospitalization Most often No
Indwelling Catheters Often No Infection Risk High No Thrombosis
Risk High No Expense High Low Ease of Use Low High "Mobility" of
Therapy Low High Efficacy in CHF Yes Untested Dosage Regulation
Tight via Reg Expression "Mobility" refers to ease of using when
away from home (traveling, etc); Reg, Regulated (the patient takes
an oral agent in a dose that provides the desired level of
transgene expression)
[0088] In alternative embodiments, the invention provides methods
for being able to turn on and turn off paracrine-expressing nucleic
acid or gene expression easily and efficiently for tailored
treatments and insurance of optimal safety.
[0089] In alternative embodiments, the paracrine protein or
proteins expressed by the paracrine-expressing nucleic acid(s) or
gene(s) have a beneficial or favorable effects (e.g., therapeutic
or prophylactic) on a tissue or an organ, e.g., the heart, blood
vessels, lungs, kidneys, or other targets, even though secreted
into the blood or general circulation at a distance (e.g.,
anatomically remote) from their site or sites of action.
[0090] In an exemplary embodiment of the invention, a
paracrine-expressing nucleic acid or gene encoding Urocortin-2 is
used, but other paracrine-expressing nucleic acids or genes can be
used to practice methods of this invention, including but not
limited to, e.g., for treating congestive heart failure (CHF) or
pulmonary hypertension: Urocortin-1 and Urocortin-3, Brain
Natriuretic Peptide (for CHF), Prostacyclin Synthase (for pulmonary
hypertension), Growth Hormone, and/or Insulin-like Growth Factor-1,
or any combination thereof.
[0091] In alternative embodiments the invention provides
applications, and compositions and methods, for a regulated
expression system providing for controlled expression of a
paracrine-type gene to treat a heart or lung disease, e.g.,
congestive heart failure (CHF) or pulmonary hypertension.
[0092] For example, in alternative embodiments a recombinant virus
(e.g., a long-term virus or viral vector), or a vector, or an
expression vector, and the like, can be injected, e.g., in a
systemic vein (e.g., IV), or by intramuscular (IM) injection, by
inhalation, or by a biolistic particle delivery system (e.g., a
so-called "gene gun"), e.g., as an outpatient, e.g., in a
physician's office. In alternative embodiments, days or weeks later
(e.g., four weeks later), the individual, patient or subject is
administered (e.g., inhales, is injected or swallows), a chemical
or pharmaceutical that induces expression of the
paracrine-expressing nucleic acids or genes; for example, an oral
antibiotic (e.g., doxycycline or rapamycin) is administered once
daily (or more or less often), which will activate the expression
of the gene. In alternative embodiments, after the "activation", or
inducement of expression (e.g., by an inducible promoter) of the
nucleic acid or gene, a paracrine protein is synthesized and
released into the subject's circulation (e.g., into the blood), and
subsequently has favorable physiological effects, e.g., therapeutic
or prophylactic, that benefit the individual or patient (e.g.,
benefit heart, kidney or lung function), depending on the paracrine
protein or proteins expressed. When the physician or subject
desires discontinuation of the treatment, the subject simply stops
taking the activating chemical or pharmaceutical, e.g.,
antibiotic.
[0093] The inventors have used an AAV vector encoding Urocortin-2
and administered the vector to mice using intravenous delivery. The
results showed: 1) a 17-fold increase in serum levels of the
transgene 4-6 weeks after intravenous delivery of the vector; 2)
pronounced favorable effects on cardiac contractile function
(systolic function); and 3) pronounced favorable effects on cardiac
relaxation (diastolic function).
[0094] In alternative embodiments, applications of the present
invention include: the treatment of severe, low ejection fraction
heart failure; the treatment of pulmonary hypertension; the
treatment of heart failure with preserved ejection fraction;
replacement of current therapies that require hospitalization and
sustained intravenous infusions of vasoactive peptides for the
treatment of pulmonary hypertension and heart failure; and, the
treatment of other conditions in which controlled expression of a
paracrine-type gene can be used to promote favorable effects at a
distance in the body.
Generating and Manipulating Nucleic Acids
[0095] In alternative embodiments, to practice the methods of the
invention, the invention provides isolated, synthetic and/or
recombinant nucleic acids or genes encoding paracrine polypeptides.
In alternative embodiments, to practice the methods of the
invention, the invention provides paracrine-expressing nucleic
acids or genes in recombinant form in an (e.g., spliced into) an
expression vehicle for in vivo expression, e.g., in a vector or a
recombinant virus. In other alternative embodiments, the invention
provides, e.g., isolated, synthetic and/or recombinant nucleic
acids encoding inhibitory nucleic acids (e.g., siRNA, microRNA,
antisense, ribozyme) that can inhibit the expression of genes or
messages (mRNAs) that inhibit the expression of the desired
paracrine gene.
[0096] In alternative embodiments, nucleic acids of the invention
are made, isolated and/or manipulated by, e.g., cloning and
expression of cDNA libraries, amplification of message or genomic
DNA by PCR, and the like. The nucleic acids and genes used to
practice this invention, including DNA, RNA, iRNA, antisense
nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids
thereof, can be isolated from a variety of sources, genetically
engineered, amplified, and/or expressed/generated recombinantly.
Recombinant polypeptides (e.g., paracrine chimeric proteins used to
practice this invention) generated from these nucleic acids can be
individually isolated or cloned and tested for a desired activity.
Any recombinant expression system or gene therapy delivery vehicle
can be used, including e.g., viral (e.g., AAV constructs or
hybrids) bacterial, fungal, mammalian, yeast, insect or plant cell
expression systems or expression vehicles.
[0097] Alternatively, nucleic acids used to practice this invention
can be synthesized in vitro by well-known chemical synthesis
techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc.
105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel
(1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994)
Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;
Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.
22:1859; U.S. Pat. No. 4,458,066.
[0098] Techniques for the manipulation of nucleic acids used to
practice this invention, such as, e.g., subcloning, labeling probes
(e.g., random-primer labeling using Klenow polymerase, nick
translation, amplification), sequencing, hybridization and the like
are well described in the scientific and patent literature, see,
e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND
ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT
PROTOCOLS 1N MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons,
Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND
MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I.
Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y.
(1993).
[0099] Another useful means of obtaining and manipulating nucleic
acids used to practice the methods of the invention is to clone
from genomic samples, and, if desired, screen and re-clone inserts
isolated or amplified from, e.g., genomic clones or cDNA clones.
Sources of nucleic acid used in the methods of the invention
include genomic or cDNA libraries contained in, e.g., mammalian
artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118;
6,025,155; human artificial chromosomes, see, e.g., Rosenfeld
(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);
bacterial artificial chromosomes (BAC); P1 artificial chromosomes,
see, e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors
(PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids,
recombinant viruses, phages or plasmids.
[0100] In alternative embodiments, to practice the methods of the
invention, paracrine fusion proteins and nucleic acids encoding
them are used. Any paracrine polypeptide can be used to practice
this invention, e.g., a Urocortin-1, a Urocortin-2, a Urocortin-3,
a Brain Natriuretic Peptide, a Prostacyclin Synthase, a Growth
Hormone, an Insulin-like Growth Factor-1 protein. In alternative
embodiments, the paracrine protein can be fused to a heterologous
peptide or polypeptide, such as a peptide for targeting the
polypeptide to a desired cell type, such a cardiac myocytes, or a
lung cell.
[0101] In alternative embodiments, a heterologous peptide or
polypeptide joined or fused to a protein used to practice this
invention can be an N-terminal identification peptide which imparts
a desired characteristic, such as fluorescent detection, increased
stability and/or simplified purification. Peptides and polypeptides
used to practice this invention can also be synthesized and
expressed as fusion proteins with one or more additional domains
linked thereto for, e.g., producing a more immunogenic peptide, to
more readily isolate a recombinantly synthesized peptide, to
identify and isolate antibodies and antibody-expressing B cells,
and the like. Detection and purification facilitating domains
include, e.g., metal chelating peptides such as polyhistidine
tracts and histidine-tryptophan modules that allow purification on
immobilized metals, protein A domains that allow purification on
immobilized immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle
Wash.). The inclusion of a cleavable linker sequences such as
Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between a
purification domain and the motif-comprising peptide or polypeptide
to facilitate purification. For example, an expression vector can
include an epitope-encoding nucleic acid sequence linked to six
histidine residues followed by a thioredoxin and an enterokinase
cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797;
Dobeli (1998) Protein Expr. Purif. 12:404-414). The histidine
residues facilitate detection and purification while the
enterokinase cleavage site provides a means for purifying the
epitope from the remainder of the fusion protein. Technology
pertaining to vectors encoding fusion proteins and application of
fusion proteins are well described in the scientific and patent
literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
[0102] Nucleic acids or nucleic acid sequences used to practice
this invention can be an oligonucleotide, nucleotide,
polynucleotide, or to a fragment of any of these, to DNA or RNA of
genomic or synthetic origin which may be single-stranded or
double-stranded and may represent a sense or antisense strand, to
peptide nucleic acid (PNA), or to any DNA-like or RNA-like
material, natural or synthetic in origin. Compounds use to practice
this invention include "nucleic acids" or "nucleic acid sequences"
including oligonucleotide, nucleotide, polynucleotide, or any
fragment of any of these; and include DNA or RNA (e.g., mRNA, rRNA,
tRNA, iRNA) of genomic or synthetic origin which may be
single-stranded or double-stranded; and can be a sense or antisense
strand, or a peptide nucleic acid (PNA), or any DNA-like or
RNA-like material, natural or synthetic in origin, including, e.g.,
iRNA, ribonucleoproteins (e.g., e.g., double stranded iRNAs, e.g.,
iRNPs). Compounds use to practice this invention include nucleic
acids, i.e., oligonucleotides, containing known analogues of
natural nucleotides. Compounds use to practice this invention
include nucleic-acid-like structures with synthetic backbones, see
e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;
Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)
Antisense Nucleic Acid Drug Dev 6:153-156. Compounds use to
practice this invention include "oligonucleotides" including a
single stranded polydeoxynucleotide or two complementary
polydeoxynucleotide strands that may be chemically synthesized.
Compounds use to practice this invention include synthetic
oligonucleotides having no 5' phosphate, and thus will not ligate
to another oligonucleotide without adding a phosphate with an ATP
in the presence of a kinase. A synthetic oligonucleotide can ligate
to a fragment that has not been dephosphorylated.
[0103] In alternative aspects, compounds used to practice this
invention include genes or any segment of DNA involved in producing
a paracrine polypeptide (e.g., a Urocortin-1, a Urocortin-2, a
Urocortin-3, a Brain Natriuretic Peptide, a Prostacyclin Synthase,
a Growth Hormone, an Insulin-like Growth Factor-1 protein); it can
include regions preceding and following the coding region (leader
and trailer) as well as, where applicable, intervening sequences
(introns) between individual coding segments (exons). "Operably
linked" can refer to a functional relationship between two or more
nucleic acid (e.g., DNA) segments. In alternative aspects, it can
refer to the functional relationship of transcriptional regulatory
sequence to a transcribed sequence. For example, a promoter can be
operably linked to a coding sequence, such as a nucleic acid used
to practice this invention, if it stimulates or modulates the
transcription of the coding sequence in an appropriate host cell or
other expression system. In alternative aspects, promoter
transcriptional regulatory sequences can be operably linked to a
transcribed sequence where they can be physically contiguous to the
transcribed sequence, i.e., they can be cis-acting. In alternative
aspects, transcriptional regulatory sequences, such as enhancers,
need not be physically contiguous or located in close proximity to
the coding sequences whose transcription they enhance.
[0104] In alternative aspects, the invention comprises use of
"expression cassettes" comprising a nucleotide sequences used to
practice this invention, which can be capable of affecting
expression of the nucleic acid, e.g., a structural gene or a
transcript (e.g., encoding a paracrine protein) in a host
compatible with such sequences. Expression cassettes can include at
least a promoter operably linked with the polypeptide coding
sequence or inhibitory sequence; and, in one aspect, with other
sequences, e.g., transcription termination signals. Additional
factors necessary or helpful in effecting expression may also be
used, e.g., enhancers.
[0105] In alternative aspects, expression cassettes used to
practice this invention also include plasmids, expression vectors,
recombinant viruses, any form of recombinant "naked DNA" vector,
and the like. In alternative aspects, a "vector" used to practice
this invention can comprise a nucleic acid that can infect,
transfect, transiently or permanently transduce a cell. In
alternative aspects, a vector used to practice this invention can
be a naked nucleic acid, or a nucleic acid complexed with protein
or lipid. In alternative aspects, vectors used to practice this
invention can comprise viral or bacterial nucleic acids and/or
proteins, and/or membranes (e.g., a cell membrane, a viral lipid
envelope, etc.). In alternative aspects, vectors used to practice
this invention can include, but are not limited to replicons (e.g.,
RNA replicons, bacteriophages) to which fragments of DNA may be
attached and become replicated. Vectors thus include, but are not
limited to RNA, autonomous self-replicating circular or linear DNA
or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat.
No. 5,217,879), and can include both the expression and
non-expression plasmids. In alternative aspects, the vector used to
practice this invention can be stably replicated by the cells
during mitosis as an autonomous structure, or can be incorporated
within the host's genome.
[0106] In alternative aspects, "promoters" used to practice this
invention include all sequences capable of driving transcription of
a coding sequence in a cell, e.g., a mammalian cell such as a
heart, lung, muscle, nerve or brain cell. Thus, promoters used in
the constructs of the invention include cis-acting transcriptional
control elements and regulatory sequences that are involved in
regulating or modulating the timing and/or rate of transcription of
a gene. For example, a promoter used to practice this invention can
be a cis-acting transcriptional control element, including an
enhancer, a promoter, a transcription terminator, an origin of
replication, a chromosomal integration sequence, 5' and 3'
untranslated regions, or an intronic sequence, which are involved
in transcriptional regulation. These cis-acting sequences typically
interact with proteins or other biomolecules to carry out (turn
on/off, regulate, modulate, etc.) transcription.
[0107] In alternative embodiments, "constitutive" promoters used to
practice this invention can be those that drive expression
continuously under most environmental conditions and states of
development or cell differentiation. In alternative embodiments,
"Inducible" or "regulatable" promoters used to practice this
invention can direct expression of the nucleic acid of the
invention under the influence of environmental conditions,
administered chemical agents, or developmental conditions.
Gene Therapy and Gene Delivery Vehicles
[0108] In alternative embodiments, methods of the invention
comprise use of nucleic acid (e.g., gene or polypeptide encoding
nucleic acid) delivery systems to deliver a payload of a
paracrine-encoding nucleic acid or gene, or a paracrine
polypeptide-expressing nucleic acid, transcript or message, to a
cell or cells in vitro, ex vivo, or in vivo, e.g., as gene therapy
delivery vehicles.
[0109] In alternative embodiments, expression vehicle, vector,
recombinant virus, or equivalents used to practice methods of the
invention are or comprise: an adeno-associated virus (AAV), a
lentiviral vector or an adenovirus vector; an AAV serotype AAV5,
AAV6, AAV8 or AAV9; a rhesus-derived AAV, or the rhesus-derived AAV
AAVrh.10hCLN2; an organ-tropic AAV, or a cardiotropic AAV, or a
cardiotropic AAVM41 mutant; and/or an AAV capsid mutant or AAV
hybrid serotype. In alternative embodiments, the AAV is engineered
to increase efficiency in targeting a specific cell type that is
non-permissive to a wild type (wt) AAV and/or to improve efficacy
in infecting only a cell type of interest. In alternative
embodiments, the hybrid AAV is retargeted or engineered as a hybrid
serotype by one or more modifications comprising: 1) a
transcapsidation, 2) adsorption of a bi-specific antibody to a
capsid surface, 3) engineering a mosaic capsid, and/or 4)
engineering a chimeric capsid. It is well known in the art how to
engineer an adeno-associated virus (AAV) capsid in order to
increase efficiency in targeting specific cell types that are
non-permissive to wild type (wt) viruses and to improve efficacy in
infecting only the cell type of interest; see e.g., Wu et al., Mol.
Ther. 2006 September; 14(3):316-27. Epub 2006 Jul. 7; Choi, et al.,
Curr. Gene Ther. 2005 June; 5(3):299-310.
[0110] For example, the rhesus-derived AAV AAVrh.10hCLN2 or
equivalents thereof can be used, wherein the rhesus-derived AAV may
not be inhibited by any pre-existing immunity in a human; see e.g.,
Sondhi, et al., Hum Gene Ther. Methods. 2012 October; 23(5):324-35,
Epub 2012 Nov. 6; Sondhi, et al., Hum Gene Ther. Methods. 2012 Oct.
17; teaching that direct administration of AAVrh.10hCLN2 to the CNS
of rats and non-human primates at doses scalable to humans has an
acceptable safety profile and mediates significant payload
expression in the CNS.
[0111] Also, for example, AAV vectors specifically designed for
cardiac gene transfer (a cardiotropic AAV) can be used, e.g., the
AAVM41 mutant having improved transduction efficiency and
specificity in the myocardium, see, e.g., Yang, et al. Virol J.
2013 Feb. 11; 10(1):50.
[0112] Because adeno-associated viruses (AAVs) are common infective
agents of primates, and as such, healthy primates carry a large
pool of AAV-specific neutralizing antibodies (NAbs) which inhibit
AAV-mediated gene transfer therapeutic strategies, the methods of
the invention comprise screening of patient candidates for
AAV-specific NAbs prior to treatment, especially with the
frequently used AAV8 capsid component, to facilitate individualized
treatment design and enhance therapeutic efficacy; see, e.g., Sun,
et al., J. Immunol. Methods. 2013 Jan. 31; 387(1-2):114-20, Epub
2012 Oct. 11.
Kits and Instructions
[0113] The invention provides kits comprising compositions and
methods of the invention, including instructions for use thereof.
As such, kits, cells, expression vehicles (e.g., recombinant
viruses, vectors) and the like can also be provided.
[0114] For example, in alternative embodiments, the invention
provides kits comprising compositions used to practice this
invention, e.g., comprising a urocortin-2 (UCn-2) peptide or
polypeptide; or a paracrine-encoding nucleic acid, (b) a liquid or
aqueous formulation of the invention, or (c) the vesicle, liposome,
nanoparticle or nanolipid particle of the invention. In one aspect,
the kit further comprising instructions for practicing any methods
of the invention, e.g., in vitro or ex vivo methods for increasing
a desired paracrine level in the bloodstream, or for protecting a
cell, e.g., a cardiac or lung cell; or for treating, preventing or
ameliorating diabetes or pre-diabetes.
Formulations
[0115] In alternative embodiments, the invention provides
compositions and methods for use in increasing paracrine levels in
vivo. In alternative embodiments, these compositions comprise
paracrine-encoding nucleic acids formulated for these purposes,
e.g., expression vehicles or paracrine-encoding nucleic acids
formulated in a buffer, in a saline solution, in a powder, an
emulsion, in a vesicle, in a liposome, in a nanoparticle, in a
nanolipoparticle and the like.
[0116] In alternative embodiments, the invention provides methods
comprising administration of urocortin-2 (UCn-2) peptides or
polypeptides, or UCn-2-encoding nucleic acids, to treat, ameliorate
or prevent a diabetes (including Type 1 and Type 2, or adult onset
diabetes) or pre-diabetes, or obesity or excess weight; or to
stimulate weight loss, or to act as an appetite suppressant.
Accordingly, the invention provides the appropriate formulations
and dosages of urocortin-2 (UCn-2) peptides or polypeptides, or
UCn-2-encoding nucleic acids, for same.
[0117] In alternative embodiments, the compositions (including
formulations of urocortin-2 (UCn-2) peptides or polypeptides, or
paracrine-encoding (e.g., UCn-2-encoding) nucleic acids, can be
formulated in any way and can be applied in a variety of
concentrations and forms depending on the desired in vitro, in vivo
or ex vivo conditions, including a desired in vivo or ex vivo
method of administration and the like. Details on techniques for in
vitro, in vivo or ex vivo formulations and administrations are well
described in the scientific and patent literature.
[0118] Formulations and/or carriers of the paracrine-encoding
nucleic acids, or urocortin-2 (UCn-2) peptides or polypeptides,
used to practice this invention are well known in the art.
Formulations and/or carriers used to practice this invention can be
in forms such as tablets, pills, powders, capsules, liquids, gels,
syrups, slurries, suspensions, etc., suitable for in vivo or ex
vivo applications.
[0119] In alternative embodiments, paracrine-encoding nucleic
acids, or urocortin-2 (UCn-2) peptides or polypeptides, used to
practice this invention can be in admixture with an aqueous and/or
buffer solution or as an aqueous and/or buffered suspension, e.g.,
including a suspending agent, such as sodium
carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethylene oxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
(e.g., polyoxyethylene sorbitol mono-oleate), or a condensation
product of ethylene oxide with a partial ester derived from fatty
acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan
mono-oleate). The aqueous suspension can also contain one or more
preservatives such as ethyl or n-propyl p-hydroxybenzoate.
Formulations can be adjusted for osmolarity, e.g., by use of an
appropriate buffer.
[0120] In practicing this invention, the compounds (e.g.,
formulations) of the invention can comprise a solution of
paracrine-encoding nucleic acids or genes, or urocortin-2 (UCn-2)
peptides or polypeptides, dissolved in a pharmaceutically
acceptable carrier, e.g., acceptable vehicles and solvents that can
be employed include water and Ringer's solution, an isotonic sodium
chloride. In addition, sterile fixed oils can be employed as a
solvent or suspending medium. For this purpose any fixed oil can be
employed including synthetic mono- or diglycerides, or fatty acids
such as oleic acid. In one embodiment, solutions and formulations
used to practice the invention are sterile and can be manufactured
to be generally free of undesirable matter. In one embodiment,
these solutions and formulations are sterilized by conventional,
well known sterilization techniques.
[0121] The solutions and formulations used to practice the
invention can comprise auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents, e.g., sodium acetate,
sodium chloride, potassium chloride, calcium chloride, sodium
lactate and the like. The concentration of active agent (e.g.,
paracrine-encoding nucleic acids or genes) in these formulations
can vary widely, and can be selected primarily based on fluid
volumes, viscosities and the like, in accordance with the
particular mode of in vivo or ex vivo administration selected and
the desired results, e.g., increasing in vivo paracrine
expression.
[0122] The solutions and formulations used to practice the
invention can be lyophilized; for example, the invention provides a
stable lyophilized formulation comprising paracrine-encoding
nucleic acids or genes, or urocortin-2 (UCn-2) peptides or
polypeptides. In one aspect, this formulation is made by
lyophilizing a solution comprising a paracrine-encoding nucleic
acid or gene, or urocortin-2 (UCn-2) peptides or polypeptides, and
a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose
or mixtures thereof A process for preparing a stable lyophilized
formulation can include lyophilizing a solution about 2.5 mg/mL
protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium
citrate buffer having a pH greater than 5.5 but less than 6.5. See,
e.g., U.S. patent app. no. 20040028670.
[0123] The compositions and formulations of the invention can be
delivered by the use of liposomes (see also discussion, below). By
using liposomes, particularly where the liposome surface carries
ligands specific for target cells, or are otherwise preferentially
directed to a specific tissue or organ type, one can focus the
delivery of the active agent into a target cells in an in vivo or
ex vivo application.
Nanoparticles, Nanolipoparticles and Liposomes
[0124] The invention also provides nanoparticles,
nanolipoparticles, vesicles and liposomal membranes comprising
compounds (e.g., paracrine-encoding nucleic acids or genes, or
urocortin-2 (UCn-2) peptides or polypeptides) used to practice the
methods of this invention, e.g., to deliver paracrine-encoding
nucleic acids or genes, or urocortin-2 (UCn-2) peptides or
polypeptides, to an individual, a patient or mammalian cells in
vivo or ex vivo. In alternative embodiments, these compositions are
designed to target specific molecules, including biologic
molecules, such as polypeptides, including cell surface
polypeptides, e.g., for targeting a desired cell type, e.g., a
mammalian cardiac cell, a kidney cell, a lung cell, a nerve cell
and the like.
[0125] The invention provides multilayered liposomes comprising
compounds used to practice this invention, e.g., as described in
Park, et al., U.S. Pat. Pub. No. 20070082042. The multilayered
liposomes can be prepared using a mixture of oil-phase components
comprising squalane, sterols, ceramides, neutral lipids or oils,
fatty acids and lecithins, to about 200 to 5000 nm in particle
size, e.g., to entrap a paracrine-encoding nucleic acid or
gene.
[0126] Liposomes can be made using any method, e.g., as described
in Park, et al., U.S. Pat. Pub. No. 20070042031, including method
of producing a liposome by encapsulating an active agent (e.g.,
paracrine-encoding nucleic acids or genes, or urocortin-2 (UCn-2)
peptides or polypeptides), the method comprising providing an
aqueous solution in a first reservoir; providing an organic lipid
solution in a second reservoir, and then mixing the aqueous
solution with the organic lipid solution in a first mixing region
to produce a liposome solution, where the organic lipid solution
mixes with the aqueous solution to substantially instantaneously
produce a liposome encapsulating the active agent; and immediately
then mixing the liposome solution with a buffer solution to produce
a diluted liposome solution.
[0127] In one embodiment, liposome compositions used to practice
this invention comprise a substituted ammonium and/or polyanions,
e.g., for targeting delivery of a compound (e.g.,
paracrine-encoding nucleic acids or genes) used to practice this
invention to a desired cell type, as described e.g., in U.S. Pat.
Pub. No. 20070110798.
[0128] The invention also provides nanoparticles comprising
compounds (e.g., paracrine-encoding nucleic acids or genes, or
urocortin-2 (UCn-2) peptides or polypeptides) used to practice this
invention in the form of active agent-containing nanoparticles
(e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat.
Pub. No. 20070077286. In one embodiment, the invention provides
nanoparticles comprising a fat-soluble active agent of this
invention or a fat-solubilized water-soluble active agent to act
with a bivalent or trivalent metal salt.
[0129] In one embodiment, solid lipid suspensions can be used to
formulate and to deliver paracrine-encoding nucleic acids or genes,
or urocortin-2 (UCn-2) peptides or polypeptides, used to practice
the invention to a patient, an individual, or mammalian cell in
vivo or ex vivo, as described, e.g., in U.S. Pat. Pub. No.
20050136121.
Delivery Vehicles
[0130] In alternative embodiments, any delivery vehicle can be used
to practice the methods or compositions of this invention, e.g., to
deliver paracrine-encoding nucleic acids or genes, or urocortin-2
(UCn-2) peptides or polypeptides, to practice the methods of the
invention in vivo or ex vivo. For example, delivery vehicles
comprising polycations, cationic polymers and/or cationic peptides,
such as polyethyleneimine derivatives, can be used e.g. as
described, e.g., in U.S. Pat. Pub. No. 20060083737.
[0131] In one embodiment, a dried polypeptide-surfactant complex is
used to formulate a composition of the invention, wherein a
surfactant is associated with a nucleic acid via a non-covalent
bond e.g. as described, e.g., in U.S. Pat. Pub. No.
20040151766.
[0132] In one embodiment, a nucleic acid or polypeptide used to
practice this invention can be applied to cells as polymeric
hydrogels or water-soluble copolymers, e.g., as described in U.S.
Pat. No. 7,413,739; for example, a nucleic acid or protein can be
polymerized through a reaction between a strong nucleophile and a
conjugated unsaturated bond or a conjugated unsaturated group, by
nucleophilic addition, wherein each precursor component comprises
at least two strong nucleophiles or at least two conjugated
unsaturated bonds or conjugated unsaturated groups.
[0133] In one embodiment, a nucleic acid or protein is applied to
cells using vehicles with cell membrane-permeant peptide
conjugates, e.g., as described in U.S. Pat. Nos. 7,306,783;
6,589,503. In one aspect, the nucleic acid itself is conjugated to
a cell membrane-permeant peptide. In one embodiment, a nucleic
acid, protein, and/or the delivery vehicle are conjugated to a
transport-mediating peptide, e.g., as described in U.S. Pat. No.
5,846,743, describing transport-mediating peptides that are highly
basic and bind to poly-phosphoinositides.
[0134] In one embodiment, electro-permeabilization is used as a
primary or adjunctive means to deliver a paracrine-encoding nucleic
acids or genes to a cell, e.g., using any electroporation system as
described e.g. in U.S. Pat. Nos. 7,109,034; 6,261,815;
5,874,268.
Products of Manufacture, Implants and Artificial Organs
[0135] The invention also provides products of manufacture
comprising cells of the invention (e.g., cells modified to express
paracrine proteins, or urocortin-2 (UCn-2) peptides or
polypeptides, to practice the methods of the invention), and use of
cells made by methods of this invention, including for example
implants and artificial organs, bioreactor systems, cell culture
systems, plates, dishes, tubes, bottles and flasks comprising cells
modified to express paracrine proteins to practice the methods of
the invention. Any implant, artificial organ, bioreactor systems,
cell culture system, cell culture plate, dish (e.g., petri dish),
cell culture tube and/or cell culture flask (e.g., a roller bottle)
can be used to practice this invention.
[0136] In alternative embodiments the invention provides a
bioreactor, implant, stent, artificial organ or similar device
comprising cells modified to express paracrine proteins to practice
the methods of the invention; for example, including implants as
described in U.S. Pat. Nos. 7,388,042; 7,381,418; 7,379,765;
7,361,332; 7,351,423; 6,886,568; 5,270,192; and U.S. Pat. App. Pub.
Nos. 20040127987; 20080119909 (describing auricular implants);
20080118549 (describing ocular implants); 20080020015 (describing a
bioactive wound dressing); 20070254005 (describing heart valve
bio-prostheses, vascular grafts, meniscus implants); 20070059335;
20060128015 (describing liver implants).
Implanting Cells In Vivo
[0137] In alternative embodiments, the methods of the invention
also comprise implanting or engrafting cells, e.g., cardiac, lung
or kidney cells, comprising or expressing paracrine-encoding
nucleic acids or genes, or urocortin-2 (UCn-2) peptides or
polypeptides, used to practice the invention; and in one aspect,
methods of the invention comprise implanting or engrafting the
paracrine-encoding nucleic acids or genes (or cells expressing
them), or urocortin-2 (UCn-2) peptides or polypeptides, in a
vessel, tissue or organ ex vivo or in vivo, or implanting or
engrafting the re-programmed differentiated cell in an individual
in need thereof.
[0138] Cells can be removed from an individual, treated using the
compositions and/or methods of this invention, and reinserted
(e.g., injected or engrafted) into a tissue, organ or into the
individual, using any known technique or protocol. For example,
de-differentiated re-programmed cells, or re-programmed
differentiated cells, can be re-implanted (e.g., injected or
engrafted) using microspheres e.g., as described in U.S. Pat. No.
7,442,389; e.g., in one aspect, the cell carrier comprises a
bulking agent comprising round and smooth polymethylmethacrylate
microparticles preloaded within a mixing and delivery system and an
autologous carrier comprising these cells. In another embodiment,
the cells are readministered to a tissue, an organ and/or an
individual in need thereof in a biocompatible crosslinked matrix,
as described e.g., in U.S. Pat. App. Pub. No. 20050027070.
[0139] In another embodiment, the cells of the invention (e.g.,
cells made by practicing the methods of this invention) are
readministered (e.g., injected or engrafted) to a tissue, an organ
and/or an individual in need thereof within, or protected by, a
biocompatible, nonimmunogenic coating, e.g., as on the surface of a
synthetic implant, e.g., as described in U.S. Pat. No. 6,969,400,
describing e.g., a protocol where a cAMP-incompetent AC can be
conjugated to a polyethylene glycol that has been modified to
contain multiple nucleophilic groups, such as primary amino or
thiol group.
[0140] In one embodiment, the cells of the invention (e.g., cells
made by practicing the methods of this invention) are
readministered (e.g., injected or engrafted) to a tissue, an organ
and/or an individual in need thereof using grafting methods as
described e.g. by U.S. Pat. Nos. 7,442,390; 5,733,542.
[0141] Any method for delivering polypeptides, nucleic acids and/or
cells to a tissue or organ (e.g., a lung, kidney, heart) can be
used, and these protocols are well known in the art, e.g., as
described in U.S. Pat. No. 7,514,401, describing e.g., using
intracoronary (IC), intravenous (IV), and/or local delivery
(myocardial injection) of polypeptides, nucleic acids and/or cells
to a heart in situ. For example, in alternative embodiments,
aerosol drug particles into the lungs and into the bloodstream,
gene therapy, continuous infusions, repeated injections and/or
sustained release polymers can be used for delivering polypeptides,
nucleic acids and/or cells to a tissue or organ (e.g., a lung,
kidney, heart). In alternative embodiments, nucleic acids and/or
cells can be given through a catheter into the coronary arteries or
by direct injection into the left atrium or ventricular myocardium
via a limited thoracotomy; or delivered into the myocardium via a
catheter passed during cardiac catheterization; or delivered into
the pericardial space.
[0142] In alternative embodiments, nucleic acids or proteins used
to practice this invention, or a vector comprising a nucleic acid
used to practice the invention (e.g., an AAV, or adenoviral gene
therapy vector), or vesicle, liposome, nanoparticle or nanolipid
particle (NLP) of the invention, and the like, to a tissue or organ
(e.g., a lung, kidney, heart); e.g. as described in U.S. Pat. No.
7,501,486, e.g., polypeptides of the invention comprising an amino
acid sequence CRPPR (SEQ ID NO:1), the amino acid sequence CARPAR
(SEQ ID NO:2) or a peptidomimetic thereof, or amino acid sequence
CPKRPR (SEQ ID NO:3) or a peptidomimetic thereof.
[0143] Compositions used to practice this invention can be used in
combination with other therapeutic agents, e.g. angiogenic agents,
anti-thrombotic agents, anti-inflammatory agents, immunosuppressive
agents, anti-arrhythmic agents, tumor necrosis factor inhibitors,
endothelin inhibitors, angiotensin-converting enzyme inhibitors,
calcium antagonists, antibiotic agents, antiviral agents and viral
vectors.
[0144] Compositions used to practice this invention can be used for
ameliorating or treating any of a variety of cardiopathies and
cardiovascular diseases, e.g., cardiopathies and cardiovascular
diseases, e.g., coronary artery disease (CAD); atherosclerosis;
thrombosis; restenosis; vasculitis including autoimmune and viral
vasculitis such as polyarteritis nodosa, Churg-Strass syndrome,
Takayasu's arteritis, Kawasaki Disease and Rickettsial vasculitis;
atherosclerotic aneurysms; myocardial hypertrophy; congenital heart
diseases (CHD); ischemic heart disease and anginas; acquired
valvular/endocardial diseases; primary myocardial diseases
including myocarditis; arrhythmias; and transplant rejections;
metabolic myocardial diseases and myocardiomyopathies such as
congestive, hypertrophic and restrictive cardiomyopathies, and/or
heart transplants. In alternative embodiments, compositions used to
practice this invention, e.g., urocortin-2 (UCn-2) peptides or
polypeptides, are used for treating, ameliorating or protecting
(preventing) diabetes or pre-diabetes in a patient or an
individual; or suppressing weight gain, or suppressing the
appetite, or stimulating or initiating weight loss, in a patient or
an individual; or treating, ameliorating or protecting (preventing)
diabetes in a patient or an individual.
[0145] The invention will be further described with reference to
the following examples; however, it is to be understood that the
invention is not limited to such examples.
EXAMPLES
Example 1
Intravenous Delivery of AAV9 Encoding Urocortin-2 Increases Cardiac
Function in Normal Mice
[0146] This example demonstrates the effectiveness of an exemplary
embodiment of the invention: intravenous delivery of
AAV9/urocortin-2 (or AAV9/UCn2) provided sustained increases in
serum UCn2 and LV contractile function, indicating the
effectiveness of this exemplary embodiment of the invention for the
treatment of heart failure.
[0147] In this study, we developed and tested the relative efficacy
of two adeno-associated virus (AAV) serotypes (AAV5 and AAV9)
encoding urocortin-2 (UCn-2), which is a vasoactive peptide in the
corticotropin-releasing factor family that has protean beneficial
effects in animals and patients with heart failure. AAV5.Ucn-2 and
AAV9.Ucn-2 (5.times.10.sup.11 genome copies, gc) were delivered by
intravenous injection (IV). Four weeks (wks) after gene transfer,
AAV DNA (qPCR) was elevated in liver (AAV5.UCn2: 2,601,839
copies/.mu.g; AAV9.UCn2: 30,121,663 copies/.mu.g) and heart (AAV5:
87,635gc/.mu.g; AAV9: 300,529 copies/.mu.g; and mRNA was similarly
elevated compared to endogenous UCn2 (AAV5.Ucn-2: 68.+-.xx-fold;
AAV9.Ucn-2: 8,575).
[0148] Left ventricular samples showed Ucn2 mRNA elevation only
with AAV9.UCn2, which was increased 28 fold over endogenous mRNA.
Plasma Ucn-2 was increased (AAV5.UCn2: from 2.7 ng/ml pre to 3.6
ng/ml, p<0.0001; AAV9.UCn2. Finally, associated with increased
serum UCn2 levels were increases in LV contractile function.
Example 2
Gene Transfer for the Treatment of Cardiovascular Diseases
[0149] This example demonstrates the effectiveness of an exemplary
embodiment of the invention in obtaining high yield transgene
expression in the heart in a manner that can be easily and safely
applied.
[0150] In alternative embodiments, the invention provides methods
using expression vehicles, e.g., vectors, encoding a paracrine-type
transgene. In this embodiment, the transgene acts as a hormone,
having cardiac effects after being released to the circulation from
a distant site. In alternative embodiments this approach can
circumvent the problem of attaining high yield cardiac gene
transfer and enable patients to be treated by a systemic injection
during an office visit.
[0151] We examined multiple AAV serotype vectors and delivery
methods, and successfully completed proof-of-concept studies of
paracrine gene transfer. Rats with severe dilated CHF underwent
skeletal muscle delivery of an adeno-associated virus 5 (AAV5)
vector encoding Insulin-like Growth Factor I (IGFI) under
tetracycline regulation. This enabled activation of IGFI expression
upon adding doxycycline in the rat's water supply. The system
provided sustained elevation of serum levels of IGFI and improved
function of the failing heart.
[0152] In alternative embodiments, a) IGFI gene transfer is used to
increase contractile function; b) AAV vectors and promoters are
used for intravenous delivery to provide maximal transgene
expression with minimal off-target effects; c) regulated transgene
expression is used to enable fine-tuning of serum transgene levels,
and allow turning expression off and on as needed; d) gene transfer
of paracrine-expressing genes, e.g., in a rat model of CHF is used;
and e) effective doses of AAV are used, and activators of transgene
expression are used following intravenous delivery of the vector,
e.g., in normal pigs, using serum paracrine (e.g., IGFI) as an
end-point.
[0153] In alternative embodiments, IV injection of an AAV vector
with regulated expression of selective peptides will, through
paracrine-mediated actions, have favorable effects on the failing
heart.
[0154] Vector Selection.
[0155] In alternative embodiments adeno-associated virus (AAV)
vectors are used, enabling long-term transgene expression superior
to adenovirus, while avoiding the potential for insertional
mutagenesis associated with lentivirus vectors. Persistent serum
elevation of Factor IX, erythropoietin and .alpha.1-antitrypsin,
have been documented in dogs and nonhuman primates, years after
single injections of AAV vectors.sup.1-4 and we have confirmed
persistent (>1 year) serum elevation of IGFI after intramuscular
injection of AAV5.IGFI-tet in rats in our laboratory..sup.5
Although recent clinical trials have found that some AAV serotypes
incite immune responses,.sup.6,7 newer generation AAV vectors do
not appear to have similar problems in preclinical studies in
primates.
[0156] AAV Serotypes: In alternative embodiments an AAV serotype
AAV2 is used, but in some embodiments, "pseudotyped" AAV vectors
are preferred. These AAV serotypes, which include AAV5, AAV6, AAV8
and AAV9, are hybrid constructs that include the capsid of AAV2 and
unique replication components that confer their specific
nomenclature. In alternative embodiments, Intravenous delivery of
AAV6, AAV8 and AAV9 is used; these show substantial distribution
and transgene expression in heart, liver, skeletal muscle, and
elsewhere.
[0157] We found intravenous better than intramuscular AAV5 in
increasing serum levels of IGFI, as illustrate in FIG. 7, which
graphically show data of free IGFI serum levels 3 months after IV
vs. IM AAV5.IGFI.tet gene transfer: Intravenous delivery in mice
(n=3, each group) provided a 2-fold increase in serum IGFI after
activation of IGFI expression with doxycycline (On); Intramuscular
delivery in rats (n=9 each group) provided a >1.3-fold increase
in serum IGFI 5 weeks after activation of IGFI expression. P values
above bars: within group comparison (t-test, 2 tails). Change in
serum IGFI was greater after intravenous delivery of AAV5.IGFI.tet
(p<0.001).
[0158] When given intravenously, AAV9 was superior to AAV5 in terms
of transgene expression in liver and heart, as illustrate in FIG. 8
graphically, and by image, illustrates data showing the relative
efficacy of intravenous delivery of exemplary AAV5 and AAV9
constructs of the invention using copy number and transgene
expression in liver and heart as endpoints, as illustrated in FIG.
8.
[0159] In alternative embodiments, AAV8, like AAV9, provides
generalized expression, but provides a higher proportion in liver
than other organs, a property that, in combination with a
liver-specific promoter, we propose to exploit.
[0160] In alternative embodiments, self-complementary AAV vectors
(scAAV) vectors are used; they can provide higher transgene
expression than their single stranded (ssAAV) analogs..sup.8
Transgene expression using the ssAAV vectors (insert capacity 4.7
kb) is delayed 4-6 w until the complementary DNA strand is
synthesized. By encoding for the complementary DNA strand within
the vector, scAAV (insert capacity 3.3 kb), enables transgene
expression in 2 w and results in higher transgene expression vs its
ssAAV analog..sup.8
[0161] Only one regulated expression vector (AAV8.TBG.IGFI.tet) may
be amenable to scAAV construction, the others are too large, as
illustrated in FIG. 10. However, if this vector is selected for the
pig studies, ssAAV can be used to provide better yield for
manufacturing the large amounts required. The scAAV analog can be
used for human use, taking advantage of superior expression,
enabling reduced dose requirements, and improving safety in the
clinical trials.
TABLE-US-00002 TABLE 2 Tetracycline vs Rapamycin Regulation Feature
Tetracycline Rapamycin Activator Doxycycline AP22594 Basal
Expression Very low/none None ("leak") Linear Dose-Response Yes Yes
Activator Side-effects Low Immunosuppressant (avoid in pregnancy)
Bacterial/Viral Proteins Yes No Used in Clinical Trials Not yet Not
yet TG, transgene; AP22594, oral rapamycin analog, 100-fold less
immune suppression vs rapamycin.sup.14
[0162] Promoter Vs Target Tissue.
[0163] In alternative embodiments, the promoter selected for
transgene expression in AAV vectors has some tissue-dependence. In
alternative embodiments, promoters used to practice the invention
include: chicken .beta.-actin (CBA); thyroid hormone-binding
globulin (TBG, liver-specific); and Rous Sarcoma Virus (RSV)
promoters. In this regard, CMV has consistently been shown to be a
superior promoter in skeletal and cardiac muscle. Recent studies
indicate that the CMV promoter is susceptible to methylation in
liver, which eventually shuts off transgene expression. Losing
liver expression would reduce serum levels of transgene--we
therefore have elected not to use the CMV promoter, selecting
instead similarly robust promoters less susceptible to methylation:
chicken .beta.-actin (CBA); thyroid hormone-binding globulin (TBG,
liver-specific); and Rous Sarcoma Virus (RSV) promoters, as
illustrated in FIG. 10.
[0164] Regulated Expression. In alternative embodiments, using
long-term expression conferred by AAV-mediated gene transfer,
transgene expression is regulated to turn off expression if
unexpected untoward effects are seen. Regulated expression would
also enable intermittent rather than constant delivery. In
alternative embodiments, the system can be configured so that the
activator either turns off or turns on transgene expression. In
instances where nearly constant transgene expression is required,
an "Off" system is desirable (e.g., one takes the oral activator
only when transgene expression is not desired, for example in the
event of adverse effects). In alternative embodiments, in instances
where intermittent transgene expression is required, an "On" system
is desirable (e.g., one takes the oral activator only for those
times that transgene expression is desired).
[0165] These alternative embodiments enable tight control, and,
when tailored for the specific disease treated, provide a means to
take the least amount of activator. In alternative embodiments,
regulated expression systems are used, e.g., ecdysone, tamoxifen,
tetracycline, rapamycin.sup.9-12; the large size of the ecdysone
system may require a two-vector strategy that would be difficult to
develop for clinical gene transfer because of regulatory
constraints. The tamoxifen system, while not as cumbersome,
requires a less tolerated activator than the tetracycline system
(tamoxifen vs doxycycline). In alternative embodiments, only two of
the available options (tetracycline and rapamycin regulation) may
be suitable, and these are the only systems that have been tested
in large animal models..sup.3,4 Both of these systems possess
analogous features (Table 2, above): the gene of interest is
controlled by an engineered transcription factor inducible by an
activating drug (tetracycline or a rapamycin analog).
[0166] Tetracycline-Regulated Expression. In alternative
embodiments the invention uses tetracycline-regulated expression in
the setting of gene transfer:
[0167] a) Basal expression of transgene ("leak"). Newer rtTA
variants, such as the one we propose and have used in recent
studies (rtTA2.sup.S-M2), provide robust tetracycline-dependent
expression with no basal activity,.sup.13 unlike previous rtTA
constructs.
[0168] b) Chronic use of tetracycline vis-a-vis patient
tolerability and off target effects. [0169] The tet-regulation
system has been extensively studied;.sup.11 in vitro studies show
that doxycycline-stimulated transgene expression begins at 0.001
ng/ml and reaches a maximum at 0.1 .mu.g/ml, a 10-fold reduction in
EC.sub.50 vs the first generation system..sup.13 In humans, a
single oral dose of 200 mg doxycycline provides mean plasma and
tissue concentrations of 1.5 .mu.g/ml at 24 h,.sup.14 15-fold
higher than required for maximal expression. A single daily dose of
doxycycline of 10-20 mg may suffice for complete activation of
transgene expression in human subjects..sup.15 Doses of 200 mg/d
are well-tolerated by patients using oral doxycycline chronically
for acne and chronic infections..sup.14,16 [0170] ORACEA.RTM.
(doxycycline 40 mg orally once daily) is approved by the FDA for
continuous use to treat rosacea..sup.16 This dose, 80% lower than
the 200 mg dose required to treat infection, provides
anti-inflammatory effects that treat rosacea, but does not have
anti-microbial effects, and does not lead to the development of
antibiotic-resistant organisms (11 years of clinical data). Each
capsule contains 40 mg of anhydrous doxycycline as 30 mg of
immediate-release and 10 mg of delayed-release beads. Subjects with
allergies to tetracycline, increased photosensitivity, pregnant or
lactating women, or children less than 9 years old (discoloration
of teeth, possible reduced long bone growth) should not use
doxycycline. In 5 years of clinical use, the most common side
effect was mild gastrointestinal complaints..sup.16 [0171]
Tetracyclines may attenuate matrix metalloproteinase expression and
activity, and have an impact on left ventricular (LV) remodeling
when administered in the first few days after myocardial infarction
(MI)..sup.17 However, in the proposed preclinical studies,
doxycycline is administered 5 weeks after MI, when LV chamber
dilation and scar formation are stable and equal among groups. We
have previously documented that doxycycline does not influence LV
remodeling, TIMP, or MMP expression in the proposed murine
MI-induced CHF model..sup.18 In the clinical setting, tetracycline
will not be used in the acute phase of MI.
[0172] c) Immune responses to the components of the rtTA system
Immune responses to the tet-regulator were not identified in a
long-term study that used AAV4.tet and AAV5.tet gene transfer
(intra-retinal) in non-human primates,.sup.3,15 where they saw
sustained tetracycline-dependent transgene expression for the 2.5
year duration of the study. We do not see inflammation in mouse
hearts expressing high levels of rtTA,.sup.18,19 or in mice and
rats after AAV5-mediated regulated expression of IGFI using the
rtTA2.sup.S-M2 regulation element..sup.5 It appears that
intramuscular delivery of AAV.tet in nonhuman primates, unlike
intra-retinal or vascular delivery, does lead to attenuation of
regulated expression, owing to immune responses to the bacterial
and virus component of the transactivator fusion protein..sup.20
Immune response to the tet-regulator can be and simultaneously, the
rapamycin-regulation system, which does not possess bacterial or
virus proteins, and is not associated with provocation of the
immune response, can be determined..sup.7 See Table 2 for strengths
and limitations of tet- and rapamycin regulation.
[0173] Rapamycin-Regulated Expression. In alternative embodiments,
a macrolide sirolimus (rapamycin), a product of the bacterium
Streptomyces hygroscopicus, is used: it was initially developed as
an anti-fungal agent, but was found to have anti-proliferative and
immunosuppressant effects. Currently it is used clinically: a) to
prevent rejection in organ transplantation (2 mg P.O. (per os,
orally), once daily, which provides mean serum levels of 12.+-.6
ng/ml); and b) in drug-eluting stents to reduce restenosis after
angioplasty, owing to its antiproliferative effects. Rapamycin
increases life span in mice, appears to forestall deleterious
effects of aging,.sup.21 and is used as an adjuvant in the
treatment of glioblastoma multiforme..sup.22 Rapamycin binds
cytosolic FK-binding protein 12 (FKBP12) and inhibits the mammalian
target of rapamycin (mTOR) signaling pathway. A serine/threonine
protein kinase, mTOR influences cell growth and proliferation, and
promotes cell survival. Rapamycin's usefulness vis-a-vis gene
therapy lies in its dimerization properties, a feature that is
exploited in the rapamycin-regulated expression system. In this
system, the DNA-binding and activation domains of an engineered
transcription factor are expressed separately as fusion proteins,
which are cross linked, and thereby activated, by the addition of a
bivalent "dimerizing" drug, in this case rapamycin or a rapamycin
analog..sup.12 Expression is dose-dependent, reversible, and is
triggered by nanomolar concentrations of activator..sup.12 The
rapamycin system contains no virus or bacterial proteins, and is
therefore unlikely to incite an immune response. In macaques,
intramuscular injection of AAV1 encoding erythropoietin provided up
to 6 year (yr) Rap-regulated expression (26 separate induction
cycles) with no decline in levels of erythropoietin, and no immune
response to the regulation elements..sup.4 Immunosuppression is a
potential disadvantage to rapamycin. However, this problem can be
circumvented by using an oral rapamycin analog (AP22594), which
activates transgene expression as effectively as rapamycin,
exhibits minimal immune suppression, and does not inhibit
mTOR..sup.4 Furthermore, as an activator, weekly rather than daily
doses are effective, further reducing off-target effects. The
dose-response relationship of orally administered AP22594, and its
maximal dose-intervals, starting with oral doses used effectively
in macaques (0.45 mg, once weekly), can be determined in
pigs..sup.4
Insulin-Like Growth Factor I (IGFI)
[0174] Selection of IGFI. Growth hormone (GH) exerts many of its
effects through activation of IGFI. IGFI exerts many of its effects
through Akt. Because of the convergence of signaling from GH
through IGFI to Akt, the selection of IGFI over GH or Akt must be
defended. Increased GH expression would be predicted to increase
serum glucose and blood pressure--deleterious effects that are
avoided by selecting IGFI. Increased expression of Akt would be
expected to reduce apoptosis, but have other potentially favorable
effects not provided by Akt, such as increased angiogenesis. We
therefore selected IGFI gene transfer for our initial preclinical
CHF studies, and recently shown that IGFI gene transfer improves
function of the failing rat heart.sup.5 (see FIGS. 1-8 and Tables 4
& 5).
[0175] IGFI Signaling. IGFs, initially known as somatomedins, are a
family of peptides that mediate many of the anabolic and mitogenic
activities of GH. Two somatomedins with structural and metabolic
similarities to insulin were isolated from human plasma in 1978 and
named IGFI and IGFII. IGFI (somatomedin C) subsequently was shown
to be the IGF regulated by circulating GH. IGFI has 70 amino acids
in a single chain with 3 disulfide bridges and a molecular weight
of 7.6 kD. Initially thought to be generated only by the liver, it
has been shown to be produced by many tissues, including intestine,
brain, kidney, lung and heart. Liver-specific deletion of the IGFI
gene in rats does not alter normal growth and development,.sup.23
indicating that IGFI, expressed widely in other tissues including
heart, regulates growth and development through local tissue
release in a paracrine manner.
[0176] IGFI belongs to a family of proteins including ligands
(IGFI, IGFII, insulin), six known binding proteins (IGFBP 1-6), and
cell surface receptors including IGFI and insulin receptors..sup.24
IGFI is translated as a pre-pro peptide which includes an amino
terminal signal peptide, A, B, C and D domains and a variable
carboxyl terminal E peptide. There are three known isoforms of
pro-IGFI in humans (pro-IGFIa, pro-IGFIb and pro-IGFIc) that differ
only in the amino acid composition of the variable E peptide. IGF
binding proteins (IGFBP) act as carrier proteins and prolong the
half-life of IGF by inhibiting degradation..sup.24 Almost all (98%)
of IGFI circulates bound predominantly (80%) to IGFBP-3..sup.24
[0177] IGFI and IGFII display high affinity binding to the IGFI
receptor in all tissues except liver. The IGF receptor shares 60%
homology with the insulin receptor and contains a tyrosine kinase
domain. Receptor binding of IGFI results in autophosphorylation of
tyrosine residues. This activates the receptor, producing
phosphorylation of substrates including the insulin receptor
substrate, which activates multiple signaling cascades including
the PI3 kinase/Akt and mitogen activated protein kinase (MAPK)
pathways, and others, many of which have beneficial cardiovascular
effects (see below Sections and Table 3).
[0178] Effects of Increased IGFI. Increased serum IGFI lowers serum
insulin levels, increases insulin sensitivity and improves lipid
profiles..sup.24 However, infusion of IGFI protein can cause
hypotension and hypoglycemia..sup.25 GH, which opposes insulin
activity, increases serum glucose levels. The ability of IGFI to
increase glucose uptake in the heart may play a role in post
ischemic recovery of LV function after IGFI administration. IGFI
increases muscle blood flow and has vasodilator activity, through
receptor dependent and independent effects and nitric oxide
production..sup.26 The combined metabolic and vasodilator effects
of high-dose intravenous IGFI infusion in humans may cause
lightheadedness and flushing--lower doses increase cardiac
performance, do not affect blood pressure or serum glucose, and are
unassociated with symptoms..sup.25,27
[0179] IGFI receptor activation is responsible for numerous
cellular responses including regulation of gene expression,
stimulation of myogenesis, cell cycle progression, immune
modulation, and steroidogenesis. In the heart, IGFI and the IGFI
receptor/PI3K/Akt signaling pathway have beneficial effects on
cardiac myocyte function, growth and survival. Moreover, IGF
exhibits angiogenic effects,.sup.28 increases cardiac contractile
function in normal .sup.25,29,30 and failing
hearts,.sup.27,29,30-33 and inhibits apoptosis..sup.34,35,38 These
features make IGFI attractive for CHF therapy (Table 3).
TABLE-US-00003 TABLE 3 IGFI: Beneficial Cardiovascular Effects
Feature Mechanism Species Ref SVR Vasodilation via NO R, P, H 25,
26, 32, 39* LV dP/dt, Inotrope; vasodilation R, D, H 25, 29, 30, 36
EF or CO LV Function Inotrope, R, D, P 27, 29-33, 39* in CHF
Ca.sup.2+ handling Cardiac Apoptosis via Akt M, R 34, 35, 38
Protection LV Mass CM prolif; M, R 31, 36, 45, 46 Apotosis via Akt
Blood flow Angiogenesis R 28 SVR, systemic vascular resistance; CO,
cardiac output; EF, ejection fraction; LV, left ventricular;
prolif, proliferation; M, mouse; R, rat; D, dog; P, pig; H, human;
39* used GH, which elevated IGFI 2-fold
[0180] IGFI Protein in Treatment of Heart Disease (Table 3)
[0181] Preclinical Studies. The effects of administering
recombinant human IGFI or GH protein in animal models of heart
diseases have been studied. IGFI is a positive inotrope in isolated
rat hearts and ferret papillary muscle; GH has no inotropic effect
in the same tissues..sup.29 Similar inotropic effects of IGFI were
found in isolated papillary muscles from dogs with pacing induced
heart failure..sup.30 IGFI administered to normal rats for four
weeks increased cardiac function and resulted in concentric LV
hypertrophy..sup.31 IGFI and GH administered together for two weeks
was associated with increased LV dP/dt and LV hypertrophy in normal
rats..sup.36 Administration of IGFI prior to myocardial ischemia
and reperfusion in rats decreased creatine kinase release and
reduced apoptosis..sup.34 Combined IGFI and GH.sup.32 or IGFI
alone.sup.33 administered four weeks after MI increased LV function
in rats. GH given to rats for four weeks after MI increased LV
systolic function,.sup.37 reduced cardiac fibrosis, cardiac myocyte
apoptosis, and increased survival..sup.38 In the pacing model of
CHF in pigs, GH increased serum IGFI, increased LV function and
reduced LV wall stress..sup.39
[0182] Clinical Studies. Clinical use of GH or IGFI protein has
received considerable attention, although there is a paucity of
large placebo-controlled studies. The acute hemodynamic effects of
IGFI infusion were studied in a blinded placebo-controlled
crossover study of CHF patients (n=8). Four-hour infusions of IGFI
increased cardiac output, decreased vascular resistance, and
reduced right atrial and wedge pressures..sup.27 Chronic
administration of IGFI protein has not been evaluated in patients
with CHF. Use of GH protein in patients with CHF has produced
equivocal results. Two small uncontrolled and unblinded studies in
a total of 14 patients with CHF reported that three months of GH
protein therapy increased serum IGFI, LV function and clinical
status..sup.40,41 Randomized placebo-controlled trials of GH
(protein) given for up to 3 months in patients with CHF did not
alter LV function or clinical status..sup.42,43 The most recent
literature review of GH protein therapy concludes that evidence for
efficacy in ischemic and idiopathic clinical CHF is lacking,
perhaps due to the kinetics of peptide administration..sup.44 Thus,
in alternative embodiments, the gene transfer methods of this
invention, by providing sustained IGFI expression, can be superior
to IGFI protein therapy.
[0183] Increased Expression of Cardiac IGFI or GH. Cardiac-directed
expression of human IGFI in rats, with its attendant increase in
cardiac myocyte IGFI production, nearly doubles serum IGFI levels.
These rats have increased heart weights with cardiac myocyte
hyperplasia, but no increase in cardiac myocyte volume..sup.35,45
After MI, reduced cardiac myocyte apoptosis, and increased
phosphorylation of Akt were found..sup.35 Cardiac-directed
expression of IGFI attenuates age-related cell senescence with
reductions in telomerase activity, telomere shortening and DNA
damage. These rats show increased Akt activation, and increased LV
function at 22 months of age vs age-matched transgene negative
littermates..sup.46 Co-expression of cardiac IGFI in a
cardiomyopathic background (crossbreeding paradigm) appears to
prevent cardiac apoptosis, LV remodeling and LV dysfunction..sup.47
However, since CHF was never present, this strategy is not
equivalent to treating already existing CHF, an approach that is a
central theme in the current proposal.
[0184] To determine if GH gene transfer would influence LV
remodeling after MI, rat cardiac muscle was directly injected with
adenovirus encoding GH (Ad.GH) at the time of coronary
occlusion..sup.48 Injections were made in the border zone between
jeopardized and viable myocardium. Six weeks after MI and gene
transfer, favorable effects were seen on LV end-diastolic
dimension, LV dP/dt and wall thickness in the infarct region. The
same scientists subsequently showed that Ad.GH injected into the
infarct border zone of rats three weeks after coronary artery
occlusion increased LV dP/dt and attenuated LV dilation and wall
thinning three weeks after injection..sup.49 GH gene transfer
during or 3 w after MI appeared to have beneficial effects on LV
remodeling.
[0185] When adenovirus encoding IGFI (Ad.IGFI) was injected into
the jeopardized perfusion bed just before coronary occlusion in
rats, the extent of infarction was reduced 50%, an effect thought
primarily to be the result of reduced apoptosis..sup.50 This study
did not address the effects of IGFI gene transfer on LV remodeling
after MI. Adenovirus mediated gene transfer of IGFI has been shown
to reduce hypoxia-induced myocyte apoptosis in vitro, and, in a rat
ischemia reperfusion model, prior injection of adenovirus encoding
IGFI reduced infarct size approximately 50% (p<0.003), although
the transgene was expressed in only about 15% of the ischemic
region, consistent with a regional paracrine effect. The effect of
expressing IGFI in the globally failing heart has not been
explored.
[0186] Potential IGFI Adverse Effects
[0187] Survival. Disruption of the GH/IGFI system appears to
increase, not decrease longevity in rats with normal cardiac
function..sup.51 However, we propose to increase IGFI expression in
the setting of severe CHF, which portends markedly increased
short-term mortality. No data suggest that IGF inhibition increases
longevity in CHF. To the contrary, increased serum IGFI in humans
reduces the incidence of CHF and mortality..sup.52,53
Epidemiological studies have shown that people with low serum IGFI
are at increased risk of developing ischemic heart disease. In the
Framingham study, individuals above the median value for serum IGFI
had a 50% reduced incidence of CHF compared to those below the
median..sup.52,53 A recent report shows that angiotensin converting
enzyme inhibitors (ACEI), which prolong life in CHF, increases IGFI
signaling..sup.54 Our data show that IGFI gene transfer increases
function of the failing rat heart, and we propose to determine
whether there also is a survival benefit.
[0188] Cancer. Clinical epidemiological studies report a
correlation between increases in serum IGFI levels (>2-fold
elevations) and prostate and premenopausal breast cancer,.sup.55
but there is no indication that this correlation is causal. It is
noteworthy that the incidence of prostate cancer increases with
age, while serum IGFI concentration decreases..sup.55 In cancer
patients, increased serum IGFI may originate in the tumor. Indeed,
increased expression of IGFI in prostate epithelium of rats
elevates serum IGFI concentrations and can lead to prostate
neoplasia..sup.56 Increased serum IGFI concentrations may also
result from changes in nutritional status in cancer patients. One
could speculate that IGFI may increase tumor growth through
angiogenesis and reduced apoptosis. Cardiac-directed expression of
IGFIb, with attendant sustained elevations in serum IGFI, is not
associated with prostate or breast cancer and combined increases in
serum IGFI and GH do not increase the incidence of prostate, breast
or lung cancer in patients with acromegaly..sup.54 The role of IGFI
in the genesis or progression of cancer is theoretical. It seems
prudent that therapies that increase IGFI expression should limit
serum concentrations of IGFI, and also provide a means to stop
expression if desired. We propose to achieve these goals by using
gene transfer of a regulated expression vector, which increases
IGFI concentrations in the serum and thereby has beneficial
cardiovascular effects.
[0189] Novelty of Studies. These studies are focused on the
development of IGFI gene transfer for clinical CHF. IGFI (or GH)
gene transfer has not been used in clinical CHF. No double-blinded
placebo-controlled clinical trial of GH/IGFI protein in CHF has
been successful, perhaps due to the relatively short biological
half-life of GH/IGFI protein, a problem that would be overcome by
gene transfer. Although GH and IGFI cardiac gene transfer have been
used prior to coronary occlusion to reduce infarct size in animal
studies, no previous study has examined IGFI gene transfer for CHF
per se. In addition, the proposed paracrine approach using systemic
delivery of a long term and regulated expression vector is new, and
can be applied to other paracrine-based peptides to treat a variety
of cardiovascular diseases.
[0190] Summary. Because of the limitations of preclinical and
clinical studies vis-a-vis predictable benefits of peptide
administration of IGFI in the treatment of severe CHF, and the
theoretical promise of paracrine-based gene transfer of IGFI, we
embarked on studies in our laboratory (see Preliminary Data),
designed to circumvent impediments and shortcomings of continuous
or chronic intermittent intravenous peptide infusion.
[0191] Other Beneficial Peptides.
[0192] Although the use of IGFI is compelling, it should be
emphasized that the paracrine gene therapy methods of the invention
are also suited for any circulating peptide with beneficial
cardiovascular effects. For example, urocortin-2 is a recently
discovered vasoactive peptide in the corticotropin-releasing factor
family that acts via corticotropin-releasing factor type 2
receptors, which are robustly expressed in the heart and
vasculature. Infusions of urocortin-2 peptide have protean
beneficial effects in animals and patients with heart
failure..sup.57 BNP is another biologically effective peptide for
the treatment of clinical CHF that could be delivered in a similar
manner. Moreover, in pulmonary hypertension, prostacyclin analogs
can be effective in treating pulmonary hypertension, but current
agents (epoprostenol and trepostinil) require constant systemic
injection, and the treatment itself is associated with high
morbidity..sup.58 In alternative embodiments, methods of the
invention provide a regulated expression vector encoding
prostacyclin synthase as a paracrine-type gene therapy of pulmonary
hypertension. Indeed, any current peptide therapeutic that requires
prolonged or chronic intermittent intravenous infusion, would lend
itself to this hormone-like gene transfer approach.
[0193] AAV & Immune Response in Clinical Studies. Long-term
transgene expression after intramuscular or intravascular delivery
of AAV vectors has been the rule rather than the exception in
rodents. However, studies in patients have been bedeviled by
limited expression due to immune responses to the transgene and, at
times, the AAV vector per se..sup.6 Two conclusions emerge from
these and other studies. 1) Intramuscular (as compared to
intravascular) AAV delivery generally provokes increased immune
response to the transgene and AAV capsid; and 2) success in
rodents, due to their relative immune tolerance, does not always
predict success in humans. Rodent and pig studies can be designed
with humans in mind: [0194] AAV serotypes (AAV8 and AAV9) can be
selected that are least likely to be associated with pre-existing
neutralizing antibodies in human subjects..sup.59 For example, AAV8
is associated with the lowest prevalence of anti-AAV neutralizing
antibodies (19% vs 59% for AAV1 and 50% for AAV2). Moreover, among
the minority of human subjects with AAV8/9 antibodies, 75-90% of
those subjects possess low titers, making AAV8 and AAV9 the current
optimal choices vis-a-vis anticipated immune response..sup.59 Human
sera possesses almost no seropositivity to rhesus-derived AAV
vectors, such as AAVrh.32.33,.sup.60 providing an alternative
vector if AAV8 and AAV9 prove unsuitable, although preclinical and
clinical experience with AAVrh.32.33 is limited. [0195]
Intramuscular injection of AAV vectors can be avoided because they
may incite immune responses in larger animals..sup.6 [0196] Two
species-specific IGFI proteins can be used: rat and pig. Both rat
and porcine IGFI can be used. The use of species-specific IGFI will
reduce immune responses to the transgene. Clinical trials can be
performed with the optimal vector encoding human IGFI.
[0197] Intravenous delivery of AAV8 and AAV9 is appealing because
of its simplicity, and because it is likely to achieve the highest
serum levels of therapeutic transgene at the lowest possible AAV
dose. Although seroprevalence to these AAV vectors is important in
pigs and primates, including humans, it has not been an important
factor in rodents. Preliminary sampling of pigs from our vendor
show no evidence of AAV8 or AAV9 antibodies in 7 of the 9 pigs
tested.
[0198] In alternative embodiments, expression of a transgene of the
invention is limited to a single organ, e.g., if such a strategy
provides therapeutic serum levels of that transgene. For example,
an exemplary vector of the invention is AAV8 with a
hepatocyte-specific promoter (TBG, human thyroid hormone-binding
globulin).
Paracrine-Based Gene Transfer Using IGFI.
[0199] Although we selected IGFI for these proof-of-concept
studies, in alternative embodiments, the invention comprises use of
any of the candidate genes outlined herein, and any of these genes
would be effective for the intended effect. For example, the
invention provides methods and compositions that effectively
deliver any paracrine polypeptide, e.g., a mammalian cardiotonic
peptide, a Serelaxin, a Relaxin-2, a Urocortin-2, a Urocortin-1, a
Urocortin-3, a Brain Natriuretic Peptide, a Prostacyclin Synthase,
a Growth Hormone, an Insulin-like Growth Factor-1, or any
combination thereof or, a human Urocortin-2, a Urocortin-1, a
Urocortin-3, a Brain Natriuretic Peptide, a Prostacyclin Synthase,
a Growth Hormone, an Insulin-like Growth Factor-1, or any
combination thereof.
[0200] We engineered an exemplary AAV5 vector encoding rat IGFI
(type A) that is under control of a tetracycline response element
(TRE): FIG. 1 illustrates an exemplary construct of the invention
comprising AAV5 encoding IGF 1; this exemplary AAV5 vector provides
regulated expression of IGFI: ITR, inverted terminal repeat; TRE,
tetracycline response element; IGFIAU1, Insulin-like Growth
Factor-I; SVpA, polyA from SV40 viral genome (bidirectional);
rtTA2.sup.SM2, reverse tetracycline controlled transactivator; CMV,
human cytomegalovirus early gene promoter. Total insert size, 2823
bp, fits into a scAAV5 vector (capacity 3.3 kb).
[0201] The coding sequence includes a signal peptide to ensure
extracellular secretion of IGFI. We have used this vector
(AAV5.IGFI.tet) in gene transfer experiments in cultured cardiac
myocytes: FIG. 2A illustrates data from studies where cultured
neonatal rat cardiac myocytes were infected with AAV5.IGFI.tet
(10,000 gc/cell, 2 d); the gels illustrated show that IGFI
expression was induced by doxycycline (+Dox) (2 .mu.g/ml, 3 d), but
did not occur in the absence of doxycycline (-Dox). IGFI was
detected in media by anti-AU1 antibody by immunoblotting. FIG. 2B
illustrates data where in the same experiments cardiac myocytes
were lysed in Akt lysis buffer (10 min, 4.degree. C.) and
centrifuged (12,000.times.g, 10 min); total Akt and phospho-Akt
were detected by anti-Akt and anti-phospho-T308-Akt antibodies.
IGFI expression was associated with Akt activation. After
infection, transgene expression was undetectable (no "leak") until
activation with doxycycline (FIG. 2A).
[0202] Our vector (FIG. 1) contains a more recent rtTA variant
(rtTA2.sup.S-M2), which provides robust dox-dependent expression
and low or absent basal activity, unlike previous rtTA
constructs..sup.13
Regulated IGFI Expression in Cultured Cardiac Myocytes
[0203] Cultured neonatal rat cardiac myocytes underwent gene
transfer with AAV5.IGFI-tet (10.sup.4 gc/cell, 2 days). As
graphically illustrated in FIG. 3, subsequently, doxycycline (2
.mu.g/ml) was added to media, and IGFI mRNA expression was
quantified using real-time RT-PCR. Expression of IGFI mRNA was
increased (versus (vs) unstimulated) by 1.5-fold within 30 min, and
reached a peak of 14-fold elevation by 24 hrs. At 48 hrs, IGFI mRNA
was somewhat less (10-fold), reflecting doxycycline degradation. To
turn-off IGFI expression doxycycline was removed using four
sequential PBS washes ("off-wash," see FIG. 3). IGFI mRNA rapidly
decreased after doxycycline withdrawal.
Skeletal Muscle Delivery of AAV5.IGFI.Tet Improves Function of the
Failing Heart
[0204] Skeletal Muscle Gene Transfer. We initially performed
studies in murine heart failure after indirect intracoronary
delivery of AAV5.IGFI.tet (FIG. 1), finding substantial
improvements in function of the failing heart after
cardiac-targeted delivery. However, proof-of-concept studies to
demonstrate the efficacy of a paracrine-based transfer, would
require skeletal muscle delivery of the vector. For these pivotal
studies, we used intramuscular delivery of AAV5.IGFI.tet in the
tibialis anterior muscle of rats..sup.5 AAV5 was selected because
of its well-known high expression levels after IM injection in
skeletal muscle. In all instances we have found IGFI expression in
media (cell culture experiments), and long term IGFI expression in
heart (murine CHF model) and in serum (rat model after IM
injection, mouse after IV injection), and corresponding improvement
in function of failing heart..sup.5
[0205] In the rat study, we first examined the feasibility of
skeletal muscle injection of AAV5.EGFP to provide long-term
transgene expression, as illustrated in FIG. 4A: illustrating
photomicrographs showing EGFP expression in unilateral tibialis
anterior muscle 3 weeks after AAV5.EGFP gene transfer in rats.
Contralateral uninjected tibialis anterior muscle from the same
animal shows no expression of EGFP. FIG. 4B is Table 4, which
summarizes data from the echocardiography measuring the effects of
Skeletal Muscle IGFI Expression in CHF.
MI Model of CHF & Experimental Protocol
[0206] MI was induced in rats by proximal left coronary occlusion,
resulting in large transmural infarction and severe impairment of
LV function. One week after MI, rats with impaired LV function
received 2.times.10.sup.12 genome copies (gc) of AAV5.IGFI.tet in
the anterior tibialis muscle. Four weeks later (5 w after MI), rats
with LV ejection fraction (EF)<35% were randomly assigned to two
groups: one group received doxycycline in drinking water to
activate IGFI expression (IGF-On; n=10) and the other did not
receive doxycycline (IGF-Off; n=9). Ten weeks after MI (5 w after
activation of IGFI expression), LV size and function were assessed
by echocardiography and hemodynamic studies; FIG. 5 illustrates the
experimental protocol for AAV5.IGFI.tet skeletal muscle gene
transfer in CHF.
[0207] Outcome. IGF-On rats showed increased LV ejection fraction
(p=0.02) and reduced LV end-systolic dimension (p=0.03) (Table 4,
see FIG. 4B). Furthermore, LV contractile function, assessed by the
rate of pressure development (LV+dP/dt) during dobutamine infusion,
was increased after initiation of IGFI expression (p=0.001) (Table
5, next page). In addition, favorable changes in cardiac output
(p=0.007) and stroke work (p=0.003) were observed (Table 5). Serum
IGFI was increased 5 wk after transgene activation (IGF-Off:
164.+-.24 ng/ml; IGF-On: 218.+-.11 ng/ml; p=0.008; n=9 each group).
These data indicate that skeletal muscle injection of AAV5.IGFI.tet
enables tetracycline-activated expression, increases serum IGFI
levels, and improves function of the failing heart. In alternative
embodiments, less immunogenic AAV vectors can be used, and they can
be used intravenously rather than in an intramuscular injection to
circumvent inciting immune responses, and test two regulated
expression systems.
TABLE-US-00004 TABLE 5 Effects of Activation of Skeletal Muscle
IGFI Expression in CHF IGF-Off IGF-On (n = 10) (n = 10) p HR Basal
377 .+-. 42 364 .+-. 83 0.79 (beats/min) Dobutamine 373 .+-. 29 395
.+-. 10 CO Basal 10.3 .+-. 2.2 16.3 .+-. 1.8 0.007 (ml/min)
Dobutamine 15.8 .+-. 2.4 23.2 .+-. 2.8 SW Basal 1.6 .+-. 0.4 4.1
.+-. 0.6 0.003 (ml mmHg) Dobutamine 3.8 .+-. 0.8 6.4 .+-. 1.2 LV +
dP/dt Basal 4,237 .+-. 630 6,337 .+-. 687 <0.0001 (mmHg/s)
Dobutamine 6,842 .+-. 913 12,974 .+-. 1,061 LV - dP/dt Basal -3,453
.+-. 494.sup. -4,564 .+-. 409.sup. 0.030 (mmHg/s) Dobutamine -6,036
.+-. 1,197 -8,518 .+-. 1,056 Systolic Basal 104 .+-. 12 143 .+-. 11
0.011 Pressure Dobutamine 113 .+-. 9 163 .+-. 8 (mmHg) Mean
Pressure Basal 82 .+-. 13 110 .+-. 8 0.07 (mmHg) SVR Basal 7.5 .+-.
1.3 6.8 .+-. 0.5 0.23 (Wood Units) HR, heart rate; CO, cardiac
output; SW, stroke work. Data denote mean .+-. SE. Probability
values from 2-way ANOVA, showing IGFI effect. Reference 5
Cardiac Apoptosis and Fibrosis (FIG. 6)
[0208] FIG. 6 illustrates the effects of AAV5.IGFI-tet gene
transfer on cardiac apoptosis and fibrosis. FIG. 6A graphically
illustrates data from TUNEL staining that indicated that activation
of IGFI expression (IGF-On) was associated with reduced cardiac
myocyte apoptosis (p<0.0001; 2-way ANOVA), which was reduced
more in the border than remote region. FIG. 6B illustrates
picrosirius red-stained sections of the uninfarcted
intraventricular septum from IGF-Off and IGF-On rats that showed
reduced cardiac fibrosis, and collagen fractional area was reduced
(p=0.048); FIG. 6C graphically illustrates this data from the
IGF-Off and IGF-On rats.
[0209] Intravenous vs Intramuscular Delivery of AAV5.IGFI.tet. In
preliminary studies, we determined whether intravenous gene
transfer could increase circulating IGFI levels. One week after
intravenous delivery of AAV5.IGFI.tet (5.times.10.sup.10 gc per
mouse, tail vein) mice were randomly assigned to one of two groups:
one group received doxycycline in drinking water to activate IGFI
expression (IGF-On) and the other did not receive doxycycline
(IGF-Off). Since the majority of circulating IGFI is bound to IGFI
binding proteins (IGFBPs) with high affinity and is biologically
inactive, we measured free serum IGFI, the bioactive IGFI form,
which was 2-fold higher in IGF-On than in IGF-Off mice 3 months
after activation of IGFI expression (FIG. 7, next page). Using the
intramuscular AAV5.IGFI.tet (2.times.10.sup.12 gc per rat) gene
transfer strategy outlined in Section 2.2.1.2., we found a 1.3-fold
increase of free serum IGFI in IGF-On group than IGF-Off group 5
weeks after activation of IGFI expression (FIG. 7). These data
suggest that intravenous delivery of AAV5.IGFI.tet is more
effective than intramuscular delivery vis-a-vis serum IGFI
concentrations.
[0210] Moreover, an intravenous strategy is likely to circumvent
provocation of immune response, which has been observed following
intramuscular delivery of AAV..sup.6 These experiments provide
pivotal feasibility data for our studies.
Intravenous Delivery: AAV5 Vs AAV9.
[0211] We next determined the relative efficacy of intravenous
delivery of AAV5 vs AAV9, using copy number and transgene
expression in liver and heart as endpoints, as illustrated in FIG.
8. We used self-complementary (sc) AAV vectors, enabling earlier
expression vs single-strand (ss) AAV vectors. Mice received
intravenous scAAV5.CMV.EGFP or scAAV9.CMV.EGFP
(5.times.10.sup.11gc) and were killed 21 d later. PCR primers
directed to common sequences in both vectors were used to compare
AAV DNA copy number in liver and heart. In liver, AAV9 (vs AAV5)
provided 3-fold increases in both AAV DNA copies and in EGFP
expression; in heart, a 5-fold increase in AAV DNA copies and an
8-fold increase in EGFP expression were seen. These data show that,
compared to intravenous AAV5, AAV9 can provide higher serum levels
of transgene.
Methods
[0212] FIG. 10 illustrates exemplary vectors and vector designs of
the invention: Using intravenous delivery of three vectors selected
from preliminary studies and biological features, the relative
merits of widely distributed and expressed AAV8 and AAV9 (FIG.
10A), and AAV8 with a liver-specific promoter (FIG. 10B) can be
determined. The criterion for effectiveness can be serum levels of
IGFI 6 weeks (w) after delivery. An optimal AAV vector is used to
generate two regulated expression vectors (Tet and Rap), which can
be compared following intravenous delivery in rats, as illustrated
in FIGS. 10 C-F. The criterion for effectiveness can be serum
levels of IGFI, this time examined 16 w after activation of
transgene expression (20 w after delivery).
[0213] FIGS. 10A & B. AAV vectors for the initial studies in
rats to determine the best AAV serotype for subsequent studies.
These vectors encode rat IGFI (unregulated), driven by CBA (AAV8
& AAV9) or TBG (AAV8). The best of these, based on serum IGFI
levels and duration of expression, can be used to undergo
subsequent studies to determine the optimal regulation system.
[0214] FIGS. 10C-F. Candidate vectors for studies in rats to
determine the optimal regulated expression system. Using the best
AAV vector from the initial studies (above), 2 regulated expression
vectors are generated and tested: one with Tetracycline-regulation,
the other with Rapamycin-regulation. These vectors encode regulated
expression of rat IGFI, driven by RSV (AAV8 & AAV9) or TBG
(AAV8). The CBA promoter is too large for the Rapamycin-regulation
vector, so RSV is used instead. The better of these two regulation
systems is selected for generation of the optimal vector for the
subsequent studies in normal pigs, encodes for regulated expression
of porcine IGFI. ITR, inverted terminal repeat; TRE, tetracycline
response element; IGFI, Insulin-like growth factor-I; SVpA, polyA
from SV40 virus genome (bidirectional); rtTA2.sup.SM2, reverse
tetracycline controlled transactivator; SV40en, simian virus 40
enhancer; TBG Prom, thyroid hormone-binding globulin promoter; RSV
Prom, Rous sarcoma virus promoter; FRB-p6, part of FRAP, a
rapamycin interacting protein, combined with a subunit of
transcription factor NF-.kappa.B (p65); IRE, internal transcription
reentry site; ZF, zinc finger HD1 DNA binding domain; FKBP, FK506
binding protein; pA, minimal polyadenylation segment; ZBD, zinc
finger HD DNA binding domain (8 copies).
[0215] We do not anticipate that immune responses to AAV will play
an important role in rats, although such responses are important in
dogs, pigs, humans and other primates. Immune responses should be
carefully assessed. AAV biodistribution (e.g., using qPCR using
primers amplifying common sequences in all vectors) and toxicity
(e.g., using histological analysis) can be quantified.
[0216] Group Size. The primary criterion for success can be serum
level of IGFI, which has a coefficient of variation of 20%. To
detect a 30% difference in serum IGFI between groups, assuming an
.alpha. error of 0.05 and a .beta. error of 0.10, will require a
group size of n=10.
Example 3
Delivery of AAV8 Encoding Urocortin-2 Increases Cardiac
Function
[0217] This example demonstrates that in alternative embodiments of
methods of this invention a paracrine transgene acts as a hormone
and has cardiac effects after being released to the circulation
from a distant site. This exemplary approach can circumvent the
problem of attaining high yield cardiac gene transfer and enable
patients to be treated by a systemic injection during an office
visit. Furthermore, this exemplary approach can eliminate the need
for intravenous (IV) delivery of therapeutic peptides and thereby
circumvent repeated and prolonged hospital stays, high morbidity,
and enormous economic costs. In alternative embodiments, the most
suited vector to achieve these goals is the adeno-associated virus
type 8 (AAV8), which provides long term and extensive expression
after intravenous delivery in rodents, pigs, and primates.
[0218] In alternative embodiments of methods Urocortin-2, a
recently discovered corticotropin releasing factor family
vasoactive peptide, is used as a therapeutic transgene. Urocortin-2
can act via corticotropin-releasing factor type 2 receptors, which
are robustly expressed in the heart and vasculature. Studies in
animals and patients with congestive heart failure have shown
favorable hemodynamic effects of urocortin-2 peptide infusions,
including increased contractile function independent of loading,
indicating direct cardiac effects. We established that intravenous
delivery of AAV8 using the chicken .beta.-actin promoter provides
sustained high serum levels of UCn2 and increases function of the
failing mouse heart.
[0219] To select the best specific embodiments to practice this
aspect of the invention, studies in mice and pigs can be carried
out, e.g.,: a) determine regulated transgene expression to enable
fine-tuning of plasma transgene levels, and allow turning
expression off and on as needed; and b) determine the safety,
efficacy, and mechanism of action of urocortin-2 gene transfer,
using this exemplary paracrine-based approach in an art-accepted
animal model, a mouse model of CHF. Also, use of normal pigs can
determine: a) the minimally effective vector dose required to
increase serum UCn2; b) biodistribution of the vector and
transgene; and c) toxicity.
[0220] Potential advantages of paracrine gene transfer methods of
the invention over IV peptide infusion are shown in Table 1
(above). In alternative embodiments, practicing methods of the
invention allows circumvention of infection and reduced repeated
and prolonged hospital stays, thereby reducing costs. In
alternative embodiments, systemic vector delivery is an advantage
in paracrine gene transfer by providing the highest level of
expression for any given AAV dose. The potential safety and
efficacy of this approach was recently demonstrated in an early
phase gene therapy clinical trial in patients with hemophilia
B,.sup.2 a study that has restored hope in gene therapy. In
alternative embodiments, paracrine gene transfer methods of the
invention can be suited for any circulating peptide with beneficial
cardiovascular effects.
[0221] In alternative embodiments, AAV is used to enable longer
transgene expression than adenovirus, and avoid insertional
mutagenesis associated with retrovirus. Persistent transgene
expression has been shown in large animals years after a single
injection of AAV vectors..sup.6-10 We have confirmed this in
mice.sup.11 & rats. Although recent clinical trials have found
that some AAV serotypes incite immune responses after IM
injection,.sup.12,13 newer generation AAV vectors (AAV5, 6, 8 and
9) do not have similar problems in primates..sup.14 IV AAV delivery
is superior to IM vis-a-vis serum transgene levels, and AAV9 and
AAV8 are superior to AAV5.sup.15 (and unpublished data). Moreover,
pre-existing anti-AAV8 antibodies are not as prevalent in humans
(19%) as are other AAV serotypes including AAV1 & AAV2
(50-59%)..sup.16 Our data, graphically illustrated in FIG. 11,
indicate that IV AAV8 is the optimal vector and delivery route to
attain sustained increased levels of serum UCn2 for a paracrine
approach. FIG. 11 illustrates data from: IV delivery of
AAV9.CMV.UCn2 (9.CMV), AAV9.CBA.UCn2 (9.CBA) vs AAV8.CBA.UCn2
(8.CBA); where the data indicated that all vectors were associated
with substantial increases in serum UCn2 6 w later. Numbers in bars
denote sample size for each group; p value from ANOVA. ITR,
inverted terminal repeat; SVpA, polyA from SV40 viral genome; UCn2,
urocortin-2; CBA, chicken .beta.-actin promoter; CMV enhancer,
human cytomegalovirus enhancer.
[0222] Despite its robustness in striated muscle, the CMV promoter
is susceptible to methylation and inactivation in liver,.sup.17 and
our data indicate that promoters less susceptible to methylation
are superior. Indeed, although CMV provided a sustained 2-fold
increase in UCn2 after IV vector delivery, use of the chicken
.beta.-actin (CBA) promoter resulted in 15.7-fold increase in serum
UCn2, as illustrated in FIG. 11. The hepatocyte-specific thyroid
hormone-binding globulin (TBG) promoter also can be used.
[0223] In alternative embodiments, the constructs and methods of
the invention allow for regulated expression, e.g., turning off
expression. Because of the potential for long-term expression
conferred by AAV gene transfer, the ability to turn off expression
is desirable in the event that untoward effects develop. Regulated
expression also enables the flexibility of intermittent rather than
constant transgene delivery. In alternative embodiments, the
constructs and methods of the invention use regulated expression
systems such as e.g.,: ecdysone, tamoxifen, tetracycline,
rapamycin..sup.18-21 The size of the ecdysone system requires a
two-vector strategy and tamoxifen presents issues with toxicity.
Tetracycline and rapamycin regulation systems (Table 2) have been
tested in large animal models.sup.9,10,22-26.
TABLE-US-00005 TABLE 2 Tetracycline vs Rapamycin Regulation Feature
Tetracycline Rapamycin Activator Doxycycline AP22594 Basal
Expression Very low/none None ("leak") Linear Dose-Response Yes Yes
Activator Side-effects Low Immunosuppressant (avoid in pregnancy)
Bacterial/Viral Proteins Yes No Used in Clinical Trials Not yet Not
yet TG, transgene; AP22594, oral rapamycin analog, 100-fold less
immune suppression vs rapamycin.sup.11
[0224] In alternative embodiments, the constructs and methods of
the invention use a tet-regulation system, which has been
extensively studied..sup.27 Unlike previous rtTA constructs, rtTA
variants of this invention (e.g., rtTA2.sup.S-M2), provide robust
tet-dependent expression with no basal activity (i.e. no
"leak").sup.11,26,28,29,30 and 10-fold higher sensitivity to
tetracycline (maximum transgene expression activation at 0.1
.mu.g/ml)..sup.30 A single daily dose of doxycycline of 10-20 mg
may suffice for complete activation of transgene expression in
human subjects..sup.26,31 Doses of 200 mg/d are well tolerated by
patients using oral doxycycline chronically for acne and chronic
infections..sup.31,32 Tetracyclines may attenuate matrix
metalloproteinase (MMP) activity and affect LV remodeling when
administered in the first few days after MI..sup.24 We have
previously shown that doxycycline does not influence LV remodeling,
TIMP, or MMP expression in the proposed murine MI-induced CHF
model, where doxycycline is given 5 w after MI..sup.25 In clinical
settings, tetracycline will not be used in the acute phase of
MI.
[0225] Immune responses to components of the rtTA system, a
potential problem, were not identified in a study of AAV4.tet and
AAV5.tet gene transfer (intraretinal) in non-human primates,.sup.9
where tetracycline-dependent transgene expression persisted for the
2.5 year duration of the study. We do not see inflammation in mouse
hearts expressing high levels of rtTA,.sup.25,28,29 or in mice
after AAV5-mediated regulated expression of IGFI using the
rtTA2.sup.S-M2 regulation element..sup.11 It appears that IM
delivery of AAV.tet in nonhuman primates, unlike intra-retinal or
vascular delivery, does lead to attenuation of regulated
expression, owing to immune responses to the bacterial and virus
components of the transactivator fusion protein..sup.33 Immune
response to the tet-regulator and the rapamycin-regulation system,
which does not possess bacterial or virus proteins and is not
associated with provocation of the immune response, can be
simultaneously tested..sup.10 See Table 2 for strengths and
limitations of tet- & rapamycin regulation.
[0226] In the rapamycin regulation system, transgene expression is
triggered by nanomolar concentrations of rapamycin or a rapamycin
analog, which is dose-dependent and reversible..sup.21 Rapamycin is
used clinically to suppress immune response, forestalls deleterious
effects of aging in mice.sup.23 and inhibits glioblastoma
multiforme.sup.34 by blocking the mammalian target of rapamycin
(mTOR) signaling pathway..sup.35 The oral rapamycin analog AP22594,
which activates transgene expression as effectively as rapamycin,
exhibits minimal immune suppression, and does not inhibit
mTOR..sup.10,35-37 Pigs can be used to determine the dose-response
relationship of orally administered AP22594, and its required
dosing intervals, starting with oral doses similar to those used
effectively in macaques (0.45 mg/kg, once weekly)..sup.10
[0227] In alternative embodiments, the constructs and methods of
the invention express in vivo Urocortin-2, including UCn1, UCn2 and
UCn3 (38-40 amino acids (aa)), which belong to the
corticotropin-releasing factor (CRF) family. These peptides can
stimulate corticotropin-releasing factor receptors 1 and 2 (CRF1,
CRF2). UCn1 binds to CRFR1 & CRFR2, but UCn2 & Ucn3
exclusively bind CRFR2,.sup.38-41 which are expressed in cardiac
myocytes, vasculature, gut, brain and skeletal muscle..sup.42,43,44
Although UCn1 was found in LPS-induced inflammation and was
implicated in tissue permeability, .sup.45,46 UCn2's effects, which
are diverse, have been associated with favorable biological
effects, owing in part to its affinity for CRFR2. UCn2's effects
are not entirely cAMP-dependent. For example, CRFR2 desensitization
after UCn2 binding induces PI3K/Akt signaling via translocation of
.beta.-arrestin. In addition, increased ERK1/2 signaling occurs via
disassociation of G protein .beta. & .gamma.
subunits..sup.47,48 These cAMP-independent events contribute to
reduced cardiac myocyte apoptosis. Peptide infusions of UCn2 in
preclinical and clinical CHF have consistently shown favorable
effects on LV function, and reduced activation of the
sympathoadrenal axis..sup.49-51
[0228] As listed in Table 3, below, among many beneficial effects,
UCn2 infusion using methods and compositions of the invention can
increase contractile function independent of loading conditions,
indicating direct cardiac effects..sup.52 The mechanisms for
inotropic effects have not been defined. Recent studies suggest
beneficial effects on Ca.sup.2+ handling,.sup.53 action potential
duration,.sup.54 ischemia-reperfusion injury,.sup.55-52 and the
renin-aldosterone system..sup.49 The safety and efficacy of UCn2
infusion has been confirmed in large animal models of
CHF,.sup.58,59 and in normal human subjects & patients with
CHF..sup.50,51 A recent editorial promotes its use in Class 3 &
4 CHF..sup.60
TABLE-US-00006 TABLE 3 Urocortin-2: Beneficial Cardiovascular
Effects Feature Mechanism Species Ref SVR Vasodilation M, S, H 44,
57-59 via CRFR2 CO & EF Inotrope; M, S, H 44, 57-59
vasodilation Cardiac SVR and LAP M, S, H 44, 57-59 Work LV
Diastolic Lusotrope M, R, S, H 44, 52, 56 Function Diuresis RBF
& Na S, H 49, 57-59 excretion RAS LV function All of the above
M, R, S, H 44, 49, 56, 57, 59 in CHF are reported LV IR Injury
Unknown M, R 53-55 & apoptosis SVR, systemic vascular
resistance; CRFR2, corticotropin-releasing factor receptor-2; CO,
cardiac output; EF, left ventricular ejection fraction; LV, left
ventricular; LAP, left atrial pressure; RBF, renal blood flow; RAS,
renin-angiotensin system; CM, cardiac myocyte; IR,
ischemia-reperfusion; M, mouse; R, rat; S, sheep; H, human
[0229] Since plasma half-life of UCn2 is 15 min,.sup.51 chronic
infusion is required. In contrast, in alternative embodiments,
paracrine-based UCn2 gene transfer of the invention can circumvent
impediments associated with chronic peptide infusions, as noted in
Table 1, above. By expressing only species-specific UCn2 in the two
species proposed, immune responses to the transgene will be
abrogated.
[0230] Paracrine-Based Gene Transfer Proof of Concept.
[0231] We proved that paracrine gene transfer via IM injection of
AAV5 encoding Insulin-like Growth Factor-I (AAV5.IGFI) improves
function of the failing rat..sup.11 We now also have shown that IV
delivery of AAV8 encoding UCn2 not only provides sustained high
levels of serum UCn2 (>15-fold increase), but increases function
of normal and failing hearts.
[0232] Selection of AAV Vector and Promoter.
[0233] It was clear from previous published studies that IV AAV8 or
AAV9 would provide higher levels of transgene expression than other
AAV serotypes, and CMV or CBA promoters, which generally are the
most robust, would be optimal. Therefore we engineered an AAV8
& two AAV9 vectors encoding murine UCn2 driven by CMV or CBA to
determine which vector would most effectively increase serum UCn2,
as illustrated in FIG. 11. A commercially available UCn2-specific
ELISA was used. AAV9.CMV raised serum UCn2 2.3-fold, which, while
lower than the other 2 vectors, may be sufficient for a therapeutic
response. However, AAV8.CBA was associated with a 15.7-fold rise in
serum UCn2 (AAV8.CBA.UCn2: 109.+-.7 ng/ml, n=9; Control: 7.+-.1
ng/ml). Such a high level of serum UCn2 would enable reducing the
AAV8 dose. The superiority of AAV8.CBA and AAV9.CBA over AAV9.CMV
may reflect either CBA's relative robustness or CMV's
susceptibility to methylation and inactivation in liver..sup.17 We
therefore selected AAV8.CBA for additional studies.
[0234] AAV8.CBA.UCn2 Distribution & Expression after
Intravenous Delivery.
[0235] In alternative embodiments, the constructs and methods of
the invention express in vivo by a paracrine-based gene transfer
strategy UCn2, and can be used to increase serum levels of UCn2.
Alternative embodiments do not require that UCn2 expression be
present in the heart per se, because it is the effects of
circulating UCn2 and its effects on the heart and vasculature that
will provide the therapeutic effects of the transgene, effects that
do not require UCn2 expression in cardiac myocytes themselves.
[0236] Liver Expression of UCn2.
[0237] The 15.7-fold increase in serum UCn2 documented 6 w after IV
delivery of AAV8.CBA.UCn2 (5.times.10.sup.11 gc; see FIG. 11) was
associated with a time-dependent increase in UCn2 mRNA expression
in liver, as illustrated in FIG. 12, that plateaued 4-6 weeks after
delivery, which correlated well with the steady rise in serum UCn2.
FIG. 12A graphically illustrates a time course of UCn2 mRNA
expression in liver after AAV8.CBA.UCn2 (5.times.10.sup.11 gc, IV).
Liver UCn2 expression (each bar is mean value from 2 mice) reached
a plateau 4-6 weeks after delivery, which correlated with the
plateau seen with serum UCn2 (data not shown). FIG. 12B graphically
illustrates data showing UCn2 mRNA expression in LV 6 weeks (w)
after AAV8.CBA.UCn2 (5.times.10.sup.11 gc, IV). Similar high levels
of UCn2 mRNA were seen in skeletal muscle samples (data not
shown).
[0238] Cardiac Expression of UCn2.
[0239] Although cardiac expression of UCn2 is not required for the
beneficial effects of the paracrine-based gene therapies of the
invention, we documented substantial increases in UCn2 mRNA
expression in LV samples 6 w after IV delivery of AAV8.CBA.UCn2,
see FIG. 12B. In alternative embodiments, a construct of the
invention, including e.g., AAV8, including AAV8 DNA presence and
UCn2 mRNA, can be delivered to and/or expressed in any organ or
other organs, including skeletal muscle, lung, brain, kidney,
spleen, small intestine, bone marrow.
[0240] UCn2 Gene Transfer in Normal Mice.
[0241] To determine if UCn2 gene transfer increased LV function, we
delivered AAV8.UCn2 (5.times.10.sup.11 gc) or saline (control) by
intravenous (IV) delivery in normal mice. Five weeks after UCn2
gene transfer, mice underwent an invasive procedure in which Millar
catheters (1.4 F) were placed in the LV chamber to measure pressure
development. Data acquisition and analyses were blinded to group
identity. UCn2 gene transfer increased LV contractile function
(LV+dP/dt) (FIG. 13A, left); -dP/dt also was reduced, indicating
enhanced LV relaxation (FIG. 13B, right panel). No adverse effects
on LV mass, histology, or LV structure or function were detected.
FIG. 3 graphically illustrates: LV function in normal mice 6 weeks
after IV delivery of AAV8.CBA.UCn2 (vs saline-injected control
mice. FIG. 3A: LV+dp/dt; FIG. 3B. LV-dP/dt. Values represent
mean.+-.SE. Number in bars denotes group size. UCn2 gene transfer
increased both contractile function and cardiac relaxation.
[0242] UCn2 Gene Transfer in Mice with CHF.
[0243] We used proximal left coronary occlusion to induce severe
CHF in mice, a model that we have used extensively and that mimics
aspects of clinical ischemia-based CHF..sup.25 As shown in the
protocol (FIG. 12A), 3 w after coronary occlusion, we performed
echocardiography to confirm severe LV dysfunction and chamber
dilation. We then randomly assigned enrollees to receive IV
delivery of AAV8.CBA.UCn2 (5.times.10.sup.11 gc per mouse) or an
equivalent volume of saline. Five weeks after randomization, mice
underwent repeat echocardiography and measurement of LV pressure
development and decay and their first derivative, LV+dP/dt. Data
acquisition and analyses were blinded to group identity. Despite
marked LV dysfunction that was present at the time of UCn2 gene
transfer, LV fractional area change (FAC %), an ejection fraction
surrogate, was increased (FIG. 14B). UCn2 gene transfer also
increased LV systolic (LV+dP/dt) and diastolic (LV-dP/dt) function
(FIG. 14C). Peak LV+dP/dt was increased to a value that approached
normal, confirming that the proposed strategy merits development as
a novel therapy for CHF. FIGS. 14B and 14C illustrate data showing
the effects of UCn2 transfer on the failing heart: FIG. 14A: 3 w
after MI and development of CHF, mice received IV AAV8. UCn2 or
saline; 5 w after gene transfer (8 w after MI), LV function was
assessed (blinded studies); FIG. 14B. UCn2 gene transfer increased
LV fractional area change (% FAC); FIG. 14C. UCn2 gene transfer
increased LV peak+dP/dt and peak-dP/dt, indicating marked benefits
in systolic & diastolic LV function of the failing heart.
[0244] UCn2 Gene Transfer: Effects on Cardiac Ca.sup.2+
Handling
[0245] C2.5.1.UCn2 Gene Transfer Alters Expression of SERCA2a.
[0246] AAV8.CBA.UCn2 gene transfer (5.times.10.sup.11 gc, IV) was
associated with increased expression of SERCA2a mRNA and protein in
LV samples obtained from mice 4 w after gene transfer (FIG. 15).
These changes would be anticipated to promote Ca.sup.2+
availability to the myofilament, and thereby to increase both
systolic and diastolic function, as we have observed in normal and
failing hearts following UCn2 gene transfer (FIGS. 13 and 14),
providing a plausible mechanism by which UCn2 gene transfer
increases LV function. Similar effects of UCn2 peptide have been
described in isolated cardiac myocytes..sup.53
[0247] FIG. 15 illustrates data (FIG. 15A, by graph, FIG. 15B, by
immunoblot) where normal mice received IV delivery of AAV8.CBA.UCn2
(5.times.10.sup.11 gc) or saline (CON); and four weeks later, LV
samples from the UCn2 gene transfer group showed a 2-fold increase
in SERCA2a protein expression Immunoblotting signal was normalized
to TnI content. Numbers in bars denote group size. These changes in
SERCA2a expression would be anticipated to promote Ca.sup.2+
availability at the myofilament, and thereby increase LV systolic
and diastolic function.
[0248] UCn2 Gene Transfer & Ca.sup.2+ Transients.
[0249] Cardiac myocytes (CM) were isolated from mice 4 w after
AAV8.CBA.UCn2 (5.times.10.sup.11 gc, IV). Mice that had received IV
saline were used as controls. During the measurement, cardiac
myocytes from UCn2 mice were incubated with 24 nM UCn2 peptide to
mimic serum UCn2 levels in vivo. Cardiac myocytes from mice
receiving UCn2 gene transfer showed altered Ca.sup.2+ transients
with reduced t.sub.1/2, as illustrated in FIG. 16: Ca.sup.2+
transients following UCn2 gene transfer: FIG. 16A graphically
illustrates that UCn2 gene transfer increased the rate of Ca.sup.2+
decline; FIG. 16B graphically illustrates that time-to-Ca.sup.2+
transient decay was shortened in cardiac myocytes from mice that
had received UCN2 gene transfer 4 w prior. Experiments were
repeated three times. Bars denote mean+SE; numbers in bars denote
number of cardiac myocytes; numbers above bars indicate p
value.
[0250] UCn2 is Cardioprotective.
[0251] To test UCn2's effects on hypoxic injury, we treated
cultured neonatal rat cardiac myocytes with sodium azide (NaN3),
which irreversibly binds the heme cofactor in cytochrome oxidase
and inhibits mitochondrial respiration, mimicking hypoxia-induced
cytotoxicity. UCn2 treatment protected cardiac myocytes from injury
as reflected morphologically and by reduced LDH release, as
illustrated in FIG. 17. UCn2 also protects isolated cardiac
myocytes from hypoxia-reoxygenation injury (p<0.001; data not
shown). FIG. 17 shows data that UCn2 protects cultured neonatal rat
cardiac myocytes from hypoxic injury: FIG. 17A illustrates that
UCn2 (60 nM) preserves morphological normality 24 hr after
NaN.sub.3 (10 mM) treatment; FIG. 17B graphically illustrates that
UCn2 reduced LDH release after NaN.sub.3 treatment
(p<0.001).
[0252] Effects on CREB and .beta.-Catenin.
[0253] LV samples were obtained from mice 4 w after AAV8.CBA.UCn2
(5.times.10.sup.11 gc, IV). Mice that had received IV saline were
used as controls. LV samples from mice that had received UCn2 gene
transfer showed increased phosphorylation of CREB (a 3-fold
increase, p<0.01, FIG. 18A). CREB is a transcriptional factor
that enables CRE-mediated gene expression in the heart. In
addition, UCn2 gene transfer was associated with a 2-fold increase
in LV .beta.-catenin phosphorylation (p<0.0001, FIG. 18B).
Increased .beta.-catenin phosphorylation reduces .beta.-catenin
accumulation in the intercalated disks of cardiac myocytes and
thereby reduces cardiac stiffness and diastolic dysfunction. This
may contribute to our observation that UCn2 gene transfer increases
LV relaxation in normal and failing hearts. FIG. 18 graphically
illustrates that phosphorylation of both CREB (FIG. 18A) and
.beta.-catenin (FIG. 18B) was detected in LV samples 4 w after IV
delivery of UCn2.CBA.UCn2. Control mice received IV saline.
[0254] Non-Cardiac Effects of UCn2 Gene Transfer.
[0255] IV delivery of AAV8.CBA.UCn2 (5.times.10.sup.11 gc) has a
favorable effect on glucose metabolism--an anti-diabetic effect.
For example, mice that received UCn2 gene transfer are resistant to
hyperglycemia induced by high fat diet (HFD), a model of Type 2
diabetes used in preclinical studies (FIG. 19A). Reduced glucose
levels are due to increased glucose utilization as seen in glucose
tolerance testing of HFD-fed mice (FIG. 19B). FIG. 19 illustrates
data showing UCn2 affects glucose regulation. Mice received IV
delivery of AAV8.CBA.UCn2 (5.times.10.sup.11 gc, n=8) or saline
(n=8), & standard chow for 3 w. A small reduction in fasting
blood glucose was seen in the UCn2 group. Mice then received a high
fat diet (HFD) for 8 w. Hyperglycemia was seen in Controls, as
expected, but UCn2 mice maintained normal blood glucose levels.
FIG. 19B. Mice received IV delivery of AAV8.CBA.UCn2
(5.times.10.sup.11 gc, n=8) or saline (n=8) & HFD for 2 months
& glucose tolerance tests conducted. Fasted mice received
glucose (2 mg/g body weight, IP) and glucose levels measured.
Results indicate that UCn2 gene transfer promotes glucose
utilization and protects against diet-induced hyperglycemia.
[0256] FIG. 20 illustrates exemplary constructs of the invention:
Abbreviations: ITR, inverted terminal repeat; TRE, tetracycline
response element; SVpA, polyA from SV40 viral genome
(bidirectional); rtTA2SM2, reverse tetracycline controlled
transactivator; SV40en, simian virus 40 enhancer; TBG Prom, thyroid
hormone-binding globulin promoter; RSV Prom, Rous sarcoma virus
promoter; FRB-p6, part of FRAP, a rapamycin interacting protein,
combined with a subunit of transcription factor NF-.kappa.B (p65);
IRE, internal transcription reentry site; ZF, zinc finger HD1 DNA
binding domain; FKBP, FK506 binding protein; pA, minimal
polyadenylation segment; ZBD, zinc finger HD DNA binding domain (8
copies).
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[0320] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
Sequence CWU 1
1
315PRTartificial sequencesynthetic peptide 1Cys Arg Pro Pro Arg 1 5
26PRTartificial sequencesynthetic peptide 2Cys Ala Arg Pro Ala Arg
1 5 36PRTartificial sequencesynthetic peptide 3Cys Pro Lys Arg Pro
Arg 1 5
* * * * *