U.S. patent application number 16/998489 was filed with the patent office on 2021-02-18 for methods for increasing the selective efficacy of gene therapy using car peptide and heparan-sulfate mediated macropinocytosis.
The applicant listed for this patent is Vascular Biosciences. Invention is credited to David Mann.
Application Number | 20210046148 16/998489 |
Document ID | / |
Family ID | 1000005180960 |
Filed Date | 2021-02-18 |
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United States Patent
Application |
20210046148 |
Kind Code |
A1 |
Mann; David |
February 18, 2021 |
Methods for Increasing the Selective Efficacy of Gene Therapy Using
CAR Peptide and Heparan-Sulfate Mediated Macropinocytosis
Abstract
Disclosed are compositions and methods for triggering disease
selective macropinocytosis. The compositions can serve as a marker
of disease activity and as a trigger of enhanced macropinocytosis
in tissues undergoing disease remodeling such as wound healing,
cancer, PAH, inflammation, diabetes, Crohn's disease, ulcerative
colitis, ankylosing spondylitis, diseases of the endometrium,
psoriasis, irritable bowel syndrome, arthritis, fibrotic disorders,
interstitial cystitis, autoimmune diseases, asthma, acute lung
injury, and adult respiratory distress syndrome. The compositions
can also serve as a receptor for disease selective cell penetrating
peptides in the cells and extracellular matrix of diseased
tissues.
Inventors: |
Mann; David; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vascular Biosciences |
San Diego |
CA |
US |
|
|
Family ID: |
1000005180960 |
Appl. No.: |
16/998489 |
Filed: |
August 20, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15472182 |
Mar 28, 2017 |
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16998489 |
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14649455 |
Jun 3, 2015 |
9603890 |
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PCT/US13/72768 |
Dec 3, 2013 |
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15472182 |
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61732859 |
Dec 3, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
G01N 2800/321 20130101; A61K 38/08 20130101; G01N 2333/926
20130101; C12Q 1/34 20130101; G01N 33/6893 20130101; G01N 2800/12
20130101 |
International
Class: |
A61K 38/08 20060101
A61K038/08; G01N 33/68 20060101 G01N033/68; A61K 45/06 20060101
A61K045/06; C12Q 1/34 20060101 C12Q001/34 |
Claims
1. A method for diagnosing the health status of an individual
suffering from Pulmonary Arterial Hypertension (PAH), the method
comprising: a) obtaining a sample from an individual suffering from
PAH; b) measuring heparanase and heparan sulfate levels in the
sample obtained in step a); c) determining the health status of the
individual based on the heparanase and heparan sulfate levels
obtained in step b.
2. The method of claim 1, wherein the sample is selected from the
group consisting of tissue, blood and urine.
3. The method of claim 2, wherein the sample is a blood sample.
4. The method of claim 2, wherein the same is a urine sample.
5. A method for increasing the localized efficacy of gene therapy
in an individual suffering from a disease by co-administering a
therapeutically effective amount of a composition consisting of: 1)
at least one cell-penetrating peptide; and 2) at least one other
therapeutic agent, wherein at least one compound of the composition
induces increased macropinocytosis of diseased cells.
6. The method of claim 5, wherein the disease is further
characterized by tissue with increased heparanase expression.
7. The method of claim 6, wherein the disease is selected from the
group consisting of pulmonary hypertension (PAH), interstitial lung
disease, acute lung injury (ALI), acute respiratory distress
syndrome (ARDS), atherosclerosis, sepsis, septic shock, sarcoidosis
of the lung, diabetes, asthma, ankylosing spondylitis, psoriasis,
diseases of the endometrium, pulmonary manifestations of connective
tissue diseases, including systemic lupus erythematosus, rheumatoid
arthritis, scleroderma, and polymyositis, dermatomyositis,
bronchiectasis, asbestosis, berylliosis, silicosis, Histiocytosis
X, pneumotitis, smoker's lung, bronchiolitis obliterans, the
prevention of lung scarring due to tuberculosis and pulmonary
fibrosis, other fibrotic diseases such as myocardial infarction,
endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis,
retroperitoneal fibrosis, progressive massive fibrosis,
pneumoconiosis, nephrogenic systemic fibrosis, keloid,
arthrofibrosis, adhesive capsulitis, radiation fibrosis,
fibrocystic breast condition, liver cirrhosis, hepatitis, liver
fibrosis, nonalcoholic fatty liver disease, nonalcoholic
steatohepatitis, sarcoidosis of the lymph nodes, or other organs,
inflammatory bowel disease, Crohn's disease, ulcerative colitis,
primary biliary cirrhosis, pancreatitis, interstitial cystitis,
chronic obstructive pulmonary disease, pneumoconiosis, autoimmune
diseases, cancer, angiogenic diseases, wound healing, erectile
dysfunction, chronic kidney disease, infections, trauma injuries
and systemic connective tissue diseases including systemic lupus
erythematosus, rheumatoid arthritis, psoriatic arthritis, juvenile
idiopathic arthritis scleroderma, polymyositis, and
dermatomyositis.
8. The method of claim 7, wherein the disease is PAH.
9. The method of claim 5, wherein the cell-penetrating peptide is
selected from the group consisting of CAR (SEQ ID NO: 1), tCAR (SEQ
ID NO: 2) or a CAR variant with at least 60% binding affinity to
IdoA2s-GlcNS.
10. The method of claim 9, wherein the cell-penetrating peptide is
CAR.
11. The method of claim 9, wherein the cell-penetrating peptide is
tCAR.
12. The method of claim 9, wherein the cell-penetrating peptide is
a CAR variant with at least 60% binding affinity to
IdoA2s-GlcNS.
13. The method of claim 9, wherein the therapeutic agent is
selected from the group consisting of small molecules,
polypeptides, peptides, peptidomimetics, nucleic acid-molecules,
cells and viruses.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application claiming
priority to U.S. application Ser. No. 15/472,182 filed Mar. 28,
2017, which is a divisional application claiming priority from
United States National Stage application Ser. No. 14/649,455, filed
Jun. 3, 2015, now U.S. Pat. No. 9,603,890, claiming priority under
35 U.S.C. 371 from International Patent Application No.
PCT/US13/72768 filed on Dec. 3, 2013, which claims priority from
U.S. Provisional Application No. 61/732,859, filed on Dec. 3, 2012,
the contents of which are hereby incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
molecular medicine, and specifically to cell and tissue targeting
peptides.
BACKGROUND OF THE INVENTION
[0003] Cell penetrating peptides were first discovered through
efforts to describe how HIV (Human Immunodefficiency Virus) enters
cells (Frankel 1988, Green 1988). This led to the discovery of the
TAT (Trans-Activator of Transcription) protein encoded as the TAT
gene within the HIV-1 virus as the protein responsible for cell
penetration and the identification of the protein transduction
domain, YGRKKRRQRRR, as the first cell penetrating peptide capable
of entering the cell membrane (Lindsay 2002, Wadia 2003, Snyder
2004). Since the discovery of the TAT transduction domain, other
cell penetrating peptides have been discovered (Saalik 2004).
[0004] Cell penetrating peptides have proven to be a useful tool
for the delivery of proteins, small molecule drugs, antibodies, and
other therapeutic compounds into cells and are currently being
tested in clinical trials (Johnson 2011). Cell penetrating homing
peptides have proven to be even more useful in that they can
penetrate cells selectively by tissue type or areas of disease
(Ruoslahti 2000, Kaplan 2005, Nishimura 2008).
[0005] While most cell penetrating peptides have been conjugated or
electrostatically bound to the desired therapeutic payload (Niesner
2002, Brooks 2005), recent experiments have described the ability
of a disease homing, cell-penetrating peptide CARSKNKDC (CAR) to
enhance the ability of co-administered vasodilators (fasudil,
Y-27632, imatinib, and sildenafil) to selectively lower pulmonary
pressure in animal models of pulmonary arterial hypertension (PAH)
(PCT/US11/26535) and additional experiments by Jarvinen and
Ruoslahti have described CAR's ability by itself to promote wound
healing (U.S. patent application Ser. No. 13/406,699).
[0006] However, the mechanism by which CAR enables the selective
uptake of co-administered drugs and promotes wound healing has not
been previously described. Furthermore the receptor to which CAR
homes to is yet to be identified.
[0007] There is a need to understand the mechanism by which CAR
works, and the receptor to which it binds. The benefits of
identifying the mechanism are numerous, including developing novel
diagnostic preparations used to aid in the development of new
treatments for a variety of diseases. Identification of the
receptor and understanding the related mechanism of action will
lead to novel disease applications of cell penetrating
peptides.
SUMMARY OF THE INVENTION
[0008] Heparan sulfate is a sulfated polysaccharide that is found
on the cell surface and extracellular matrix of all human cells. It
interacts with a variety of proteins and is thus involved in
numerous biological processes such as growth and development.
Heparan sulfate's function critically depends on the number and the
position of sulfate groups, which modulate the binding sites for
proteins such as growth factors, cytokines, receptors, enzymes, and
inhibitors. However, little is known about their roles both in
vitro and in vivo.
[0009] Disclosed herein is a heparan sulfate moiety,
2-O-sulfo-.alpha.-L-iduronic
acid-2-deoxy-2-acetamido-.alpha.-D-glucopyranosyl (IdoA2S-GlcNS),
capable of triggering disease selective macropinocytosis. The
moiety disclosed herein can serve as a marker of disease activity
and as a trigger of enhanced macropinocytosis in tissues undergoing
disease remodeling such as wound healing, cancer, PAH,
inflammation, diabetes, Crohn's disease, ulcerative colitis,
ankylosing spondylitis, diseases of the endometrium, psoriasis,
irritable bowel syndrome, arthritis, fibrotic disorders,
interstitial cystitis, autoimmune diseases, asthma, acute lung
injury, and adult respiratory distress syndrome. The moiety
disclosed herein can also serve as a receptor for disease selective
cell penetrating peptides in the cells and extracellular matrix of
diseased tissues.
[0010] Also disclosed herein is a sulfotransferase enzyme, Heparan
sulfate 2-O-sulfotransferase-1 (HS2ST1), involved in the synthesis
of IdoA2S-GlcNS. The sulfotransferase enzyme disclosed herein can
serve as a marker of diseases such as wound healing, cancer, PAH,
inflammation, diabetes, Crohn's disease, ulcerative colitis,
ankylosing spondylitis, diseases of the endometrium, psoriasis,
irritable bowel syndrome, arthritis, fibrotic disorders,
interstitial cystitis, autoimmune diseases, asthma, acute lung
injury, and adult respiratory distress syndrome. The
sulfotransferase enzyme disclosed herein can also serve as a marker
for the need for enhanced macropinocytosis in the tissues
expressing elevated levels of HS2ST1. Methods of treating diseases
characterized by elevated HS2ST1 are also disclosed herein.
[0011] Alterations in heparan sulfation may be associated with
pathologic conditions such as cancer. Heparan sulfate
6-O-sulfotransferases (HS6STs) catalyze the transfer of sulfate
groups to the carbon 6 position in heparan sulfate. Three isoforms
of these enzymes have been discovered in humans. Disclosed herein
is a Heparan sulfate 6-O-sulfotransferase-3, (HS6ST3), which can
serve as a marker of diseases such as wound healing, cancer, PAH,
inflammation, diabetes, Crohn's disease, ulcerative colitis,
ankylosing spondylitis, diseases of the endometrium, psoriasis,
irritable bowel syndrome, arthritis, fibrotic disorders,
interstitial cystitis, autoimmune diseases, asthma, acute lung
injury, and adult respiratory distress syndrome. The
sulfotransferase enzyme disclosed herein can also serve as a marker
for the need for enhanced macropinocytosis in the tissues
expressing elevated levels of HS6ST3. Methods of treating diseases
characterized by elevated HS6ST3 are also disclosed herein.
[0012] Also disclosed herein is a heparan sulfate degrading enzyme,
heparanase (HPSE), involved in the degradation and cleavage of
heparan sulfate moieties. The enzyme disclosed herein can serve as
a marker of diseases such as wound healing, cancer, PAH,
inflammation, diabetes, Crohn's disease, ulcerative colitis,
ankylosing spondylitis, diseases of the endometrium, psoriasis,
irritable bowel syndrome, arthritis, fibrotic disorders,
interstitial cystitis, autoimmune diseases, asthma, acute lung
injury, and adult respiratory distress syndrome. The enzyme
disclosed herein can also serve as a marker for the need for
enhanced macropinocytosis in the tissues expressing elevated levels
of HPSE. Methods of treating diseases characterized by elevated
HPSE are also disclosed herein.
[0013] Also disclosed herein are methods for improving the
selective efficacy of gene therapy. Disclosed are methods wherein
the selective efficacy of gene therapy is increased in a localized
manner for wound healing, cancer, PAH, inflammation, diabetes,
Crohn's disease, ulcerative colitis, ankylosing spondylitis,
diseases of the endometrium, psoriasis, irritable bowel syndrome,
arthritis, fibrotic disorders, interstitial cystitis, autoimmune
diseases, asthma, acute lung injury, and adult respiratory distress
syndrome. The methods of gene therapy disease selective enhancement
disclosed herein can involve co-administration with the gene
therapy and can be orally available.
[0014] Also disclosed herein are methods of treating diseases
characterized by decreased macropinocytosis by administering a
disease selective macropinocytosis promoter. The disease
characterized by decreased macropinocytosis can be wound healing,
cancer, PAH, inflammation, diabetes, Crohn's disease, ulcerative
colitis, ankylosing spondylitis, diseases of the endometrium,
psoriasis, irritable bowel syndrome, arthritis, fibrotic disorders,
interstitial cystitis, autoimmune diseases, asthma, acute lung
injury, and adult respiratory distress syndrome and the disease
selective macropinocytosis promoter can be a cell penetrating
peptide. The disease selective macropinocytosis promoters disclosed
herein can involve co-administration with the gene therapy and can
be orally available.
[0015] Additional advantages of the disclosed method and
compositions will be set forth in part in the description which
follows, and in part will be understood from the description, or
may be learned by practice of the disclosed method and
compositions. The advantages of the disclosed method and
compositions will be realized and attained by means of the elements
and combinations particularly pointed out in the appended
claims.
[0016] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the disclosed invention, and together with the
description, serve to explain the principles of the disclosed
methods.
[0018] FIG. 1 shows the effects of co-administered CAR peptide on
gene therapy. CAR peptide was able to exert its disease-selective
adjuvant properties by allowing enhanced viral delivery and uptake
of gene product (MFN2) in an animal model of severe occlusive PAH
in rats. (A) Figure showing the effect of MFN2 GTx administered
alone when compared to control and diseased animal not given MFN2
Gtx. Statistical significance was examined using ANOVA (see
Supplement for further statistical information). (B) Change in
mitofusion-2 mRNA levels relative to control. The results indicate
that MFN2 GTx is significantly enhanced in the presence of CAR
peptide. (C) The RV/LV-S ratio returned to near-normal
(non-diseased/control) levels following co-administration of CAR
with MFN2 GTx. (D) mPAP (mmHg) levels in Sugen hypoxia rats.
CAR+MFN2 GTx reduced pressure levels more than when MFN2 GTx was
administered alone.
[0019] Adenoviruses were used as viral delivery vectors to
incorporate cDNA inserts of MFN2 into rats with severe occlusive
pulmonary hypertension induced by Sugen injection followed by
hypoxia (FIG. 1). The results indicated that when administered
alone, MFN2 gene therapy brought MFN2 mRNA gene expression levels
to within normal range (FIG. 1A). In contrast to the administration
of MFN2 GTx alone (MFN2 only), when CAR was co-administered with
MFN2 GTx,(MFN2-CAR), MFN2 mRNA expression levels were significantly
elevated, greater than 7.times. above both control levels and MFN2
Gtx levels of MFN2 expression (P=0.001) (FIG. 1B). Additionally,
the ratio of right ventricle to left ventricle+septum weight ratio
(RV/LV-S ratio) returned to almost normal when CAR was
co-administered with the MFN2 GTx (FIG. 1C). Finally, Sugen hypoxia
rats treated with MFN2 GTx and CAR peptide displayed pulmonary
arterial pressure (mPAP) reductions that were significantly larger
(P<0.5) than PAH rats treated only with MFN2 adenovirus gene
therapy (FIG. 1D).
[0020] FIG. 2 shows the chemical structure of SEQ ID NO:1,
CARSKNKDC (CAR).
[0021] FIG. 3 shows CAR peptide binding and internalization
mechanism of action. FIG. 3a shows healthy glycocalyx located on
endothelial cell surface facing the bloodstream. FIG. 3b shows the
result of PAH injury. FIG. 3c shows the initiation of heparan
sulfate-mediated macropinocytosis. FIG. 3d shows vesicle formation
and CAR internalization by cells. FIG. 3e shows release of CAR into
diseased cells.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The disclosed methods and compositions can be understood
more readily by reference to the following detailed description of
particular embodiments and the Examples included therein and to the
Figures and their previous and following description.
[0023] Before the present compounds, compositions, articles,
devices, and/or methods are disclosed and described, it is to be
understood that they are not limited to specific synthetic methods
of specific recombinant biotechnology methods unless otherwise
specified, or to particular reagents unless otherwise specified, as
such may, of course, vary. It is also to be understood that the
terminology used herein is for the purposes of describing
particular embodiments only and is not intended to be limiting.
Definitions
[0024] As used in the specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a pharmaceutical carrier" includes mixtures of two or
much such carriers, and the like.
[0025] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed. It is also understood that
throughout the application, data is provided in a number of
different formats, and that this data, represents endpoints and
starting points, and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point 15 are disclosed, it is understood that greater than, greater
than or equal to, less than, less than or equal to, and equal to 10
and 15 are considered disclosed as well as between 10 and 15. It is
also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0026] In this specification and in the claims which follow,
reference will be made to a number of terms which shall be defined
to have the following meanings:
[0027] The term "bioactive agent" refers to a substance which is
used in connection with an application that is therapeutic or
diagnostic in nature, such as in methods for diagnosing the
presence or absence of a disease in a patient and/or in methods for
treating a disease in a patient. As to compatible bioactive agents,
those skilled in the art will appreciate that any therapeutic or
diagnostic agent may be incorporated in the stabilized dispersions
of the present invention. For example, the bioactive agent may be
selected from the group consisting of antiallergics,
bronchodilators, vasodilators, antihypertensive agents,
bronchoconstrictors, pulmonary lung surfactants, analgesics,
antibiotics, leukotriene inhibitors or antagonists,
anticholinergics, mast cell inhibitors, antihistamines,
anti-inflammatories, anti-neoplastics, anesthetics,
anti-tuberculars, imaging agents, cardiovascular agents, enzymes,
steroids, genetic material, viral vectors, antisense agents, small
molecule drugs, proteins, peptides and combinations thereof.
Particularly preferred bioactive agents comprise compounds which
are to be administered systemically (i.e. to the systemic
circulation of a patient) such as small molecule drugs, imaging
agents, peptides, proteins or polynucleotides. As will be disclosed
in more detail below, the bioactive agent may be incorporated,
blended in, coated on or otherwise associated with the targeting
peptide disclosed herein. Particularly preferred bioactive agents
for use in accordance with the invention include anti-allergics,
peptides and proteins, bronchodilators, anti-inflammatory agents
and anti-cancer compounds for use in the treatment of disorders
involving diseased tissue reflecting altered heparan sulfate
variants specific to said disease. Yet another associated advantage
of the present invention is the effective delivery of bioactive
agents administered or combined with a targeting peptide.
[0028] As used herein, the term "dendrimer" shall mean repeatedly
branched and roughly spherical molecules. A dendrimer is typically
symmetric around a core and usually adopts a spherical
three-dimensional morphology. Dendrimers generally contain three
major portions: a core, an inner shell and an outer shell.
Dendrimers can be synthesized to have different and varying
functionality in each of the major portions in order to control
such variables as solubility, thermal stability and attachment of
compounds suitable for particular applications.
[0029] "Optional" or "optionally" means that subsequently described
event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not.
[0030] As used herein, the phrase "other compounds and
compositions" is used broadly such that "compounds" and
"compositions" may mean a nanoparticle, a nanoworm, an iron oxide
nanoworm, an iron oxide nanoparticle, an albumin nanoparticle, a
liposome, a micelle, a phospholipid, a polymer, a microparticle, a
fluorocarbon microbubble, a therapeutic agent, a therapeutic
protein, a therapeutic compound, a therapeutic composition, a
chemotherapeutic agent, a cancer chemotherapeutic agent, a toxin, a
cytotoxic agent, Abraxane, paclitaxel, taxol, imatinib, an
anti-angiogenic agent, a pro-angiogenic agent, an anti-inflammatory
agent, an anti-arthritic agent, a TGF-B inhibitor, decorin, a
systemic vasodilator, an anti-coagulant, tissue factor pathway
inhibitor (TFPI), site-inactivated factor VIIa, a B-2 agonist,
salmeterol, formoterol, N-Acetylcysteine (NAC), Superoxide
Dismutase (SOD), a superoxide dismutase mimetic, EUK-8, an
endothelin (ET-1) receptor antagonist, a prostacyclin derivative, a
phosphodiesterase type 5 inhibitor, Ketoconazole, an antibody, a
small interfering RNA (siRNA), a microRNA (miRNA), a polypeptide, a
nucleic acid molecule, a small molecule, a carrier, a vehicle, a
virus, a phage, a viral particle, a phage particle, a viral capsid,
a phage capsid, a virus-like particle, a liposome, a micelle, a
bead, a nanoparticle, a microparticle, a detectable agent, a
contrast agent, an imaging agent, a label, a labeling agent, a
fluorophore, fluorescein, rhodamine, FAM, a radionuclide,
indium-111, technetium-99, carbon-11, or carbon-13 and the
like.
[0031] As used herein, the term "peptide" is used broadly to mean
peptides, proteins, fragments of proteins and the like. Protein
variants and derivatives are well understood by those of skill in
the art and in can involve amino acid sequence modifications. For
example, amino acid sequence modifications typically fall into one
or more of three classes: substitutional, insertional or deletional
variants. Insertions include amino and/or carboxyl terminal fusions
as well as intrasequence insertions of single or multiple amino
acid residues. Insertions ordinarily will be smaller insertions
than those of amino or carboxyl terminal fusions, for example, on
the order of one to four residues. Immunogenic fusion protein
derivatives, such as those described in the examples, are made by
fusing a polypeptide sufficiently large to confer immunogenicity to
the target sequence by cross-linking in vitro or by recombinant
cell culture transformed with DNA encoding the fusion. Deletions
are characterized by the removal of one or more amino acid residues
from the protein sequence. Typically, no more than about from 2 to
6 residues are deleted at any one site within the protein molecule.
These variants ordinarily are prepared by site specific mutagenesis
of nucleotides in the DNA encoding the protein, thereby producing
DNA encoding the variant, and thereafter expressing the DNA in
recombinant cell culture. Techniques for making substitution
mutations at predetermined sites in DNA having a known sequence are
well known, for example M13 primer mutagenesis and PCR mutagenesis.
Amino acid substitutions are typically of single residues, but can
occur at a number of different locations at once; insertions
usually will be on the order of about from 1 to 10 amino acid
residues; and deletions will range about from 1 to 30 residues.
Deletions or insertions preferably are made in adjacent pairs, i.e.
a deletion of 2 residues or insertion of 2 residues. Substitutions,
deletions, insertions or any combination thereof may be combined to
arrive at a final construct. The mutations must not place the
sequence out of reading frame and preferably will not create
complementary regions that could produce secondary mRNA structure.
Substitutional variants are those in which at least one residue has
been removed and a different residue inserted in its place.
[0032] The phrase "substantially identical" means that a relevant
sequence is at least 70%, 75%, 80%, 85%, 90%, 92%, 95% 96%, 97%,
98%, or 99% identical to a given sequence. By way of example, such
sequences may be allelic variants, sequences derived from various
species, or they may be derived from the given sequence by
truncation, deletion, amino acid substitution or addition. Percent
identity between two sequences is determined by standard alignment
algorithms such as ClustalX when the two sequences are in best
alignment according to the alignment algorithm.
[0033] A polypeptide "variant" as referred to herein means a
polypeptide substantially homologous to a native polypeptide, but
which has an amino acid sequence different from that encoded by any
of the nucleic acid sequences of the invention because of one or
more deletions, insertions or substitutions. Variants can comprise
conservatively substituted sequences, meaning that a given amino
acid residue is replaced by a residue having similar physiochemical
characteristics. (See Zubay, Biochemistry, Addison-Wesley Pub. Co.,
(1983)). It is a well-established principle of protein and peptide
chemistry that certain amino acids substitutions, entitled
"conservative" amino acid substitutions, can frequently be made in
a protein or a peptide without altering either the conformation or
the function of the protein or peptide. Such changes include
substituting any of alanine (A), isoleucine (I), valine (V), and
leucine (L) for any other of these amino acids; aspartic acid (D)
for glutamic acid (E) and vice versa; glutamine (Q) for asparagine
(N) and vice versa; serine (S) for threonine (T) and vice versa;
and arginine (R) for lysine (K) and vice versa.
[0034] In addition to the known functional variants, there are
derivatives of the peptides disclosed herein which can also
function in the disclosed methods and compositions. Protein and
peptide variants and derivatives are well understood by those of
skill in the art and in can involve amino acid sequence
modifications. For example, amino acid sequence modifications
typically fall into one or more of three classes: substitutional,
insertional or deletional variants. Insertions include amino and/or
carboxyl terminal fusions as well as intrasequence insertions of
single or multiple amino acid residues. Insertions ordinarily will
be smaller insertions than those of amino or carboxyl terminal
fusions, for example, on the order of one to four residues.
Deletions are characterized by the removal of one or more amino
acid residues from the protein or peptide sequence. Typically, no
more than about from 2 to 6 residues are deleted at any one site
within the protein or peptide molecule. These variants can be
prepared by site-specific mutagenesis of nucleotides in the DNA
encoding the protein or peptide, thereby producing DNA encoding the
variant, and thereafter expressing the DNA in recombinant cell
culture, or via solid state peptide synthesis.
[0035] Substitutional variants are those in which at least one
residue has been removed and a different residue inserted in its
place. Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative,
i.e., selecting residues that differ more significantly in their
effect on maintaining (a) the structure of the polypeptide backbone
in the area of the substitution, for example as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site or (c) the bulk of the side chain. The
substitutions which in general are expected to produce the greatest
changes in the protein properties will be those in which (a) a
hydrophilic residue, e.g. seryl or threonyl, is substituted for (or
by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,
valyl or alanyl; (b) a cysteine or proline is substituted for (or
by) any other residue; (c) a residue having an electropositive side
chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or
by) an electronegative residue, e.g., glutamyl or aspartyl; or (d)
a residue having a bulky side chain, e.g., phenylalanine, is
substituted for (or by) one not having a side chain, e.g., glycine,
in this case, (e) by increasing the number of sites for sulfation
and/or glycosylation. Similarly, the term "conformational homology"
may be used herein to define a sequence which maintains a similar
arrangement of amino acids from a conformational perspective to SEQ
ID NO:1 or SEQ ID NO:2.
[0036] It is understood that the description of conservative
mutations and homology can be combined together in any combination,
such as embodiments that have at least 70% homology to a particular
sequence wherein the variants are conservative mutations.
[0037] As this specification discusses various proteins and protein
sequences it is understood that the nucleic acids that can encode
those protein sequences are also disclosed. This would include all
degenerate sequences related to a specific protein sequence, i.e.
all nucleic acids having a sequence that encodes one particular
protein sequence as well as all nucleic acids, including degenerate
nucleic acids, encoding the disclosed variants and derivatives of
the protein sequences. Thus, while each particular nucleic acid
sequence may not be written out herein, it is understood that each
and every sequence is in fact disclosed and described herein
through the disclosed protein sequence.
[0038] It is understood that there are numerous amino acid and
peptide analogs which can be incorporated into the disclosed
compositions. For example, there are numerous D amino acids or
amino acids which have a different functional substituent than
those discussed above. The opposite stereo isomers of naturally
occurring peptides are disclosed, as well as the stereo isomers of
peptide analogs. These amino acids can readily be incorporated into
polypeptide chains by charging tRNA molecules with the amino acid
of choice and engineering genetic constructs that utilize, for
example, amber codons, to insert the analog amino acid into a
peptide chain in a site specific way (Thorson et al., Methods in
Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion in
Biotechnology, 3:348-354 (1992); Ibba, Biotechnology & Genetic
Engineering Reviews 13:197-216 (1995), Cahill et al., TIBS,
14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba
and Hennecke, Bio/technology, 12:678-682 (1994) all of which are
herein incorporated by reference at least for material related to
amino acid analogs).
[0039] Molecules can be produced that resemble peptides, but which
are not connected via a natural peptide linkage. For example,
linkages for amino acids or amino acid analogs can include
CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2-CH.sub.2-, --CH.dbd.CH-(cis
and trans), --COCH.sub.2-, --CH(OH)CH.sub.2-, and --CHH.sub.2SO--
(These and others can be found in Spatola, A. F. in Chemistry and
Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein,
eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega
Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications
(general review); Morley, Trends Pharm Sci (1980) pp. 463-468;
Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979)
(--CH.sub.2NH--, CH.sub.2CH.sub.2-); Spatola et al. Life Sci
38:1243-1249 (1986) (--CH H.sub.2-S); Hann J. Chem. Soc Perkin
Trans. I 307-314 (1982) (--CH--CH--, cis and trans); Almquist et
al. J. Med. Chem. 23:1392-1398 (1980) (--COCH.sub.2-);
Jennings-White et al. Tetrahedron Lett 23:2533 (1982)
(--COCH.sub.2-); Szelke et al. European Appln, EP 45665 CA (1982):
97:39405 (1982) (--CH(OH)CH.sub.2-); Holladay et al. Tetrahedron.
Lett 24:4401-4404 (1983) (C(OH)CH.sub.2-); and HrubyLife Sci
31:189-199 (1982) (--CH.sub.2-S--); each of which is incorporated
herein by reference. A particularly preferred non-peptide linkage
is --CH.sub.2NH--. It is understood that peptide analogs can have
more than one atom between the bond atoms, such as b-alanine,
g-aminobutyric acid, and the like.
[0040] Amino acid analogs and peptide analogs often have enhanced
or desirable properties, such as, more economical production,
greater chemical stability, enhanced pharmacological properties
(half-life, absorption, potency, efficacy, etc.), altered
specificity (e.g., a broad-spectrum of biological activities),
reduced antigenicity, and others.
[0041] D-amino acids can be used to generate more stable peptides,
because D amino acids are not recognized by peptidases and such.
Systematic substitution of one or more amino acids of a consensus
sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) can be used to generate more stable peptides.
Cysteine residues can be used to cyclize or attach two or more
peptides together. This can be beneficial to constrain peptides
into particular conformations. (Rizo and Gierasch Ann. Rev.
Biochem. 61:387 (1992), incorporated herein by reference).
[0042] Also disclosed are chimeric proteins containing a disclosed
peptide fused to a heterologous protein. In one embodiment, the
heterologous protein can have a therapeutic activity such as
cytokine activity, cytotoxic activity or pro-apoptotic activity. In
a further embodiment, the heterologous protein can be an antibody
or antigen-binding fragment thereof. In other embodiments, the
chimeric protein includes a peptide containing the amino acid
sequence SEQ ID NO: 1 or SEQ ID NO: 2, or a conservative variant or
peptidomimetic thereof, fused to a heterologous protein. The term
"heterologous," as used herein in reference to a protein fused to
the disclosed peptides, means a protein derived from a source other
than the gene encoding the peptide or from which the peptidomimetic
is derived. The disclosed chimeric proteins can have a variety of
lengths including, but not limited to, a length of less than 100
residues, less than 200 residues, less than 300 residues, less than
400 residues, less than 500 residues, less than 800 residues or
less than 1000 residues.
[0043] As used herein, "chimera" and "chimeric" refer to any
combination of sequences derived from two or more sources. This
includes, for example, from single moiety of subunit (e.g.,
nucleotide, amino acid) up to entire source sequences added,
inserted and/or substituted into other sequences. Chimeras can be,
for example, additive, where one or more portions of one sequence
are added to one or more portions of one or more other sequences;
substitutional, where one or more portions of one sequence are
substituted for one or more portions of one or more other
sequences; or a combination. "Conservative substitutional chimeras"
can be used to refer to substitutional chimeras where the source
sequences for the chimera have some structural and/or functional
relationship and where portions of sequences having similar or
analogous structure and/or function are substituted for each other.
Typical chimeric and humanized antibodies are examples of
conservative substitutional chimeras.
[0044] Also disclosed are bifunctional peptides which contain a
homing peptide fused to a second peptide having a separate
function. Such bifunctional peptides have at least two functions
conferred by different portions of the full-length molecule and
can, for example, display anti-angiogenic activity or pro-apoptotic
activity in addition to selective homing activity.
[0045] Also disclosed are isolated multivalent peptides that
include at least two subsequences each independently containing a
homing molecule (for example, the amino acid sequence SEQ ID NO: 1
or 2, or a conservative variant or peptidomimetic thereof). The
multivalent peptide can have, for example, at least three, at least
five or at least ten of such subsequences each independently
containing a homing molecule (for example, the amino acid sequence
of SEQ ID NO: 1 or 2, or a conservative variant or peptidomimetic
thereof). In particular embodiments, the multivalent peptide can
have two, three, four, five, six, seven, eight, nine, ten, fifteen
or twenty identical or non-identical subsequences. In a further
embodiment, the multivalent peptide can contain identical
subsequences, which consist of a homing molecule (for example, the
amino acid sequence SEQ ID NO: 1 or 2, or a conservative variant or
peptidomimetic thereof). In a further embodiment, the multivalent
peptide contains contiguous identical or non-identical
subsequences, which are not separated by any intervening amino
acids. In yet further embodiments, the multivalent peptide can be
cyclic or otherwise conformationally constrained. In one example,
the peptide can be circularized or cyclized via a disulfide
bond.
[0046] The term "peptidomimetic," as used herein, means a
peptide-like molecule that has the activity of the peptide upon
which it is structurally based. Such peptidomimetics include
chemically modified peptides, peptide-like molecules containing
non-naturally occurring amino acids, and peptoids and have an
activity such as selective homing activity of the peptide upon
which the peptidomimetic is derived (see, for example, Goodman and
Ro, Peptidomimetics for Drug Design, in "Burger's Medicinal
Chemistry and Drug Discovery" Vol. 1 (ed. M. E. Wolff; John Wiley
& Sons 1995), pages 803-861).
[0047] If desired, an isolated peptide, or a homing molecule as
discussed further elsewhere herein, can be cyclic or otherwise
conformationally constrained. As used herein, a "conformationally
constrained" molecule, such as a peptide, is one in which the
three-dimensional structure is maintained substantially in one
spatial arrangement over time. Conformationally constrained
molecules can have improved properties such as increased affinity,
metabolic stability, membrane permeability or solubility. Methods
of conformational constraint are well known in the art and include
cyclization as discussed further elsewhere herein.
[0048] As used herein in reference to a peptide, the term "cyclic"
means a structure including an intramolecular bond between two
non-adjacent amino acids or amino acid analogues. The cyclization
can be affected through a covalent or non-covalent bond.
Intramolecular bonds include, but are not limited to, backbone to
backbone, side-chain to backbone and side-chain to side-chain
bonds. A preferred method of cyclization is through formation of a
disulfide bond between the side-chains of non-adjacent amino acids
or amino acid analogs. Residues capable of forming a disulfide bond
include, for example, cysteine (Cys), penicillamine (Pen),
.beta.,.beta.-pentamethylene cysteine (Pmc),
.beta.,.beta.-pentamethylene-.beta.-mercaptopropionic acid (Pmp)
and functional equivalents thereof.
[0049] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to or more fully describe the state of the art
to which this pertains. The references disclosed are also
individually and specifically incorporated by reference herein for
the material contained in them that is discussed in the sentence in
which the reference is relied upon.
[0050] It is understood that the disclosed method and compositions
are not limited to specific synthetic methods, specific analytical
techniques, or to particular reagents unless otherwise specified,
and, as such, may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only and is not intended to be limiting.
[0051] Disclosed herein are peptides that enable intracellular
delivery, exit and tissue penetration of compositions. The delivery
can be targeted to cells or tissues of interest, such as tumors,
regenerating tissue, sites of injury, surgical sites, tumor
vasculature, sites of tumor angiogenesis, sites of inflammation,
sites of arthritis, lung tissue, PAH lung vasculature, PAH lesions,
remodeled pulmonary arteries, and interstitial space of lungs.
Internalization of compositions (including nanoparticles, drugs,
detectable markers, and other compounds) and their payload into
target cells and penetration into target tissue can increase the
efficiency of the targeting and the effectiveness of the
payload.
[0052] Described herein is a peptide identified as CARSKNKDC (CAR,
SEQ ID NO: 1). Also described herein is a truncated form of CAR
(CARSKNK; tCAR; SEQ ID NO: 2). It was discovered that the truncated
peptide is more potent for cell internalization and tissue
penetration than the parent peptide CAR. These properties make tCAR
a useful tool for targeted delivery of therapeutic and diagnostic
agents to breast cancers and perhaps other types of tumors as
well.
[0053] The disclosed tCAR peptides can be specific for a particular
pathological lesion or an individual tissue. Examples include
tumors, wounded tissue, diseased lung tissue, and fibrotic tissue.
The ability of compositions to penetrate into the extravascular
space is a major factor limiting the targeting efficacy of
compositions in vivo. It has been discovered that a truncated form
of the CAR homing peptide mediates highly efficient internalization
of phage and free peptides into cells.
[0054] It has also been discovered that tCAR peptides specifically
increase the penetration of drugs into tumors, wounded or injured
tissue, regenerating tissue, injured, diseased, or fibrotic lung
tissue, and other cells and tissues. Disclosed are homing peptides
that specifically increase the penetration of compounds and
compositions into vasculatures, tissues, and cells targeted by tCAR
peptides. These peptides specifically home to target tissues,
penetrate tissue, and internalize into cells. Payloads attached to
these peptides, including drug, fluorophore, nanoparticle, and the
like, accumulate in targeted tissues and penetrate deep into the
extravascular tissues, such as extravascular tumor tissues.
However, it has also been discovered that the payload does not need
to be coupled to or associated with the tCAR peptide. The free tCAR
peptide specifically induces tissue permeability in the targeted
tissues, allowing a co-injected drug, nanoparticle, and the like,
to extravasate and penetrate into the targeted tissue. This same
effect can be achieved with any cells and tissue suitable for tCAR
internalization.
[0055] The disclosed enhancement of internalization and tissue
penetration has broad application. Using the disclosed methods, the
effective targeting, delivery, and penetration of any drug,
compound or composition can be augmented and enhanced. The effect
of the disclosed methods has several significant implications.
First, drugs and other compounds and compositions can be delivered
to cells and tissues of interest at higher concentrations than is
possible in standard therapy. This is a result of the increased
internalization and tissue penetration mediated by the disclosed
peptides. This is particularly significant because the amount of
drug that can be administered is generally limited by side effects.
Increasing the drug concentration at the target without increasing
the amount of drug administered can thus extend and enhance the
effectiveness of any known or future drugs and therapeutics. When
using the disclosed methods, the increase in drug concentration
only occurs in target cells and tissues and not in non-target
tissues. In such cases, the efficacy of the treatment can be
increased, while side effects can remain the same. Second, the dose
or amount of drug or other compound or composition can be reduced
without compromising the efficacy of the treatment. The disclosed
methods would result in the same drug concentration at the target
cell or tissue even though the amount of drug administered is less.
Third, because the adjuvant peptide and the drug, imaging agent, or
other compound or composition need not be coupled to one another, a
validated and approved peptide can be used to augment any drug,
imaging agent, or other compound or composition.
[0056] It is understood that one way to define the variants and
derivatives of the disclosed proteins herein is through defining
the variants and derivatives in terms of homology/identity to
specific known sequences. For example, SEQ ID NO: 2 sets forth a
particular sequence of tCAR. Specifically disclosed are variants of
these and other peptides herein disclosed which have at least, 70%
or 75% or 80% or 85% or 90% or 95% homology to the stated sequence.
Wherein a sequence is said to have at least about 70% sequence
identity, it is understood to also have at least about 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity.
[0057] It is also understood that variants and derivatives of the
disclosed proteins herein may be defined by defining the variants
and derivatives in terms of binding affinity to specific known
sequences. For example, IdoA2S-GlcNS is a heparin sulfate moiety
that when bound by CAR triggers macropinocytosis. Specifically
disclosed are variants of CAR which have at least 60% or greater
binding affinity to IdoA2S-GlcNS. Wherein a CAR variant is said to
have at least about 60% binding affinity, it is understood to also
have about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% binding affinity.
[0058] Previously, cell-surface heparan sulfate (HS) has been shown
to be necessary for both CAR binding and internalization. When
treated with heparinase I, binding of CAR to Chinese hamster ovary
cells was greatly reduced. This suggested CAR's specific binding
and internalization is mediated by the presence of HS moieties at
the surface of the target cell. However the specific HS moiety to
which CAR was binding was not specifically identified by this
experiment.
[0059] In order to identify CAR's receptor additional information
was required. Since previous studies have described CAR homing to
pulmonary hypertensive arteries, it was deduced that the receptor
for CAR must be present in PAH. It was further deduced that
important clues to CAR's receptor could be found in PAH gene
expression data.
[0060] In a surgical shunt model of PAH, elevated gene expression
levels of HPSE, a substrate specific enzyme were found. HPSE is
responsible for the cleavage of specific HS moieties (Peterson
2010). Since high levels of HPSE were found in PAH, but CAR bound
strongly in PAH, we can conclude HPSE does not inhibit CAR binding
and furthermore, the specific HS receptor to which CAR binds is not
cleaved by HPSE. However, since heparinase I reduced CAR binding,
CAR's HS receptor is cleaved by heparinase I.
[0061] Interestingly, heparinase I, the enzyme found to inhibit CAR
binding, and HPSE do not have the same HS substrate specificity,
and one HS moiety in particular, IdoA2S-GlcNS, is cleaved by
heparinase I (Wei 2005) but not HPSE. Our PAH gene expression data
also revealed extremely elevated levels of the heparan sulfate
2-O-sulfotransferase 1 (HS2ST1) gene, which encodes the HS2ST1
enzyme and is necessary for synthesis of the IdoA2S-GlcNS HS
moiety, further suggesting that IdoA2S-GlcNS is present at elevated
levels in PAH and is likely the CAR receptor.
[0062] Since high levels of HPSE were found in PAH, it is likely
that CAR would also be useful for treating other diseases
characterized by high levels of HPSE such as tumors, chronic
inflammatory diseases, atherosclerosis, coronary artery disease,
colitis, inflammatory bowel disorders, diabetes, arterial
thrombosis, stent thrombosis, glomerular diseases, wound healing,
and endometrial disorders. CAR has already demonstrated its utility
in wound healing and homing properties to tumors further suggesting
that CAR would be useful for treating other diseases characterized
by elevated HPSE levels.
[0063] One possible mechanism by which CAR could facilitate the
selective uptake of co-administered drugs is through heparan
sulfate-mediated macropinocytosis. Macropinocytosis is a form of
endocytosis that allows for the regulated internalization of
extracellular solute molecules (Lim 2011). Studies have described
the role of heparan sulfate as the receptor for lipid
raft-dependent macropinocytotic internalization, and
macropinocytosis has also been shown to underlie the
internalization of other cationic cell-penetrating peptides (Fan
2007). Heparan sulfate mediated macropinocytosis could explain
CAR's ability to increase the localized concentration of
co-administered drugs without requiring the drugs to be conjugated
to CAR as well as CAR's ability to promote wound healing.
[0064] Heparan sulfate-mediated macropinocytosis is a non-clathrin,
non-caveolin, lipid raft-dependent form of endocytosis that allows
for the regulated internalization of extracellular solute molecules
(Lim 2011). HS-mediated macropinocytosis is utilized by various
cationic cell-penetrating peptides (Fan 2007), and is a plausible
mechanism of action to explain both CAR's ability to accelerate
wound healing as well as CAR's ability to increase the localized
concentration of co-administered drugs in diseased tissues. Wound
healing could be accelerated through CAR administration by the
selective binding of CAR to the wounded area, triggering enhanced
macropinocytosis in the wounded tissues. This accelerated
macropinocytosis would lead to an increased uptake of molecules
involved in the body's wound healing response such as cytokines and
growth factors. Similarly, the pulmonary selective vasodilation
observed in animal models of PAH when CAR is co-administered with
vasodilators could also be explained by this mechanism of action.
CAR administration and selective binding to the area of disease
would enhance HS-mediated macropinocytosis, resulting in increased
localized uptake of vasodilatory drugs in the diseased pulmonary
arteries and selective dilation of pulmonary vasculature.
[0065] CAR could also facilitate the selective treatment of other
diseases in which CAR displays homing activity alone or in
combination with other therapies by selectively increasing
macropinocytosis in diseased tissues characterized by elevated
levels of HPSE.
[0066] Diabetes, for example, is marked by elevated levels of HPSE
(Shafat 2011, Katz 2002, Ziolkowski 2012), and the HS moiety to
which CAR binds should also be present in insulin-resistant tissues
in diabetes. In this case, CAR should preferentially home to
inflamed, insulin-resistant tissue damaged by diabetes, and induce
HS-mediated macropinocytosis upon binding specifically to the
diseased tissue. This will lead to increased glucose uptake and/or
improved localized performance of insulin or other diabetes
medications at the site of inflammation and insulin resistance.
Interestingly, reduced macropinocytosis is a hallmark of
macrophages from diabetic animal models (Guest 2008). Selectively
increasing macropinocytosis in inflamed tissues associated with
diabetes would be an additional benefit of CAR administration in
diabetes.
[0067] Further evidence of the plausibility of macropinocytosis as
the mechanism of action for CAR is found in a recent experiment
combining CAR with gene therapy. Based on the demonstrated success
of CAR peptide as a disease-selective adjuvant that can be used
with various co-administered therapeutic agents, we further sought
to assess CAR's homing potential in gene therapy (GTx). Previous
efforts to increase the selectivity and delivery of gene therapy
vectors using cell penetrating peptides have required that the
peptide be covalently or electrostatically bound to the gene
therapy vector (Yao 2012). But this approach has often resulted in
suboptimal results.
[0068] The MFN2 gene encodes the mitofusion-2 protein, which is a
mitochondrial membrane protein that participates in mitochondrial
fusion. Mitofusion-2 contributes to the maintenance and operation
of the mitochondrial network, and is involved in the regulation of
vascular smooth muscle cell (VSMC) proliferation. MFN2 expression
is down-regulated in vascular proliferative disorders such as
cancer and pulmonary hypertension, and MFN2 overexpression can
attenuate the proliferation of VSMCs. Successful delivery and
uptake of the MFN2 gene via adenovirus delivery vector into
diseased cells results in increased expression of the mitofusion-2
protein, regulation of VSMC proliferation, and a subsequent
decrease in disease indicators. We hypothesized that CAR peptide
could improve the disease-selective targeting and localization of
adenoviruses containing the MFN2 gene. Better localization and
targeting of MFN2 gene therapy should result in increased MFN2 mRNA
levels, improved mitochondrial function, and a reduction in
pulmonary arterial pressure.
[0069] When we compare the results of the gene therapy experiment
with the co-administration effect we see that CAR increased
selective vasodilation approximately 2.times., and co-administered
drug concentration 2.times., but gene therapy transfected gene
expression increased 7.times.. One possible reason why gene therapy
was selectively enhanced 7.times. while drug selectivity was only
enhanced 2.times. is the mechanism of action by which adenoviruses
transfect cells through the same lipid raft dependent
macropinocytosis mechanism we are proposing for CAR's
internalization. Since CAR stimulates macropinocytosis in the
target tissues, the localized effects of gene therapy transfection
are more enhanced by the co-administration of CAR than drug
co-administration in which CAR stimulates macropinocytosis in the
target tissues to increase localized drug levels. The increased
enhancement of gene therapy versus drug co-administration further
supports lipid raft dependent macropinocytosis as CAR's mechanism
for cell penetration.
[0070] Taken together, these data identify a specific HS moiety
(IdoA2S-GlcNS) as a possible receptor for CAR that could be present
in pulmonary hypertensive tissues in which many of the HS moieties
have been cleaved by HPSE, which is over-expressed in PAH. We
further hypothesize that heparan sulfate-mediated macropinocytosis
could be a plausible mechanism by which CAR binding could trigger
the internalization of co-administered compounds and as a result
serve as a selective enhancer of gene therapy and other therapies
that utilize macropinocytosis as their route of cellular entry.
CAR's selective triggering of macropinocytosis in diseased tissues
by itself could also have therapeutic utility.
[0071] In one embodiment, the present invention discloses a peptide
that targets the receptor IdoA2s-GlcNS and selectively penetrates
the cells and extracellular matrix of diseased tissues. In a
preferred embodiment the disease is selected from the group
consisting of pulmonary hypertension, interstitial lung disease,
acute lung injury (ALI), acute respiratory distress syndrome
(ARDS), sepsis, septic shock, sarcoidosis of the lung, diabetes,
ankylosing spondylitis, psoriasis, diseases of the endometrium,
pulmonary manifestations of connective tissue diseases, including
systemic lupus erythematosus, rheumatoid arthritis, scleroderma,
and polymyositis, dermatomyositis, bronchiectasis, asbestosis,
berylliosis, silicosis, Histiocytosis X, pneumotitis, smoker's
lung, bronchiolitis obliterans, the prevention of lung scarring due
to tuberculosis and pulmonary fibrosis, other fibrotic diseases
such as myocardial infarction, endomyocardial fibrosis, mediastinal
fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive
massive fibrosis, pneumoconiosis, nephrogenic systemic fibrosis,
keloid, arthrofibrosis, adhesive capsulitis, radiation fibrosis,
fibrocystic breast condition, liver cirrhosis, hepatitis, liver
fibrosis, nonalcoholic fatty liver disease, nonalcoholic
steatohepatitis, sarcoidosis of the lymph nodes, or other organs,
inflammatory bowel disease, Crohn's disease, ulcerative colitis,
primary biliary cirrhosis, pancreatitis, interstitial cystitis,
chronic obstructive pulmonary disease, pneumoconiosis, autoimmune
diseases, angiogenic diseases, wound healing, infections, trauma
injuries and systemic connective tissue diseases including systemic
lupus erythematosus, rheumatoid arthritis, psoriatic arthritis,
juvenile idiopathic arthritis scleroderma, polymyositis, and
dermatomyositis. In another preferred embodiment, the peptide is
CAR. In another preferred embodiment, the peptide is tCAR. In yet
another preferred embodiment, the peptide is a variant of CAR with
at least 60% binding affinity to IdoA2s-GlcNS.
[0072] In one embodiment, the present invention discloses a method
of treating a disease comprising increasing macropinocytosis. In a
preferred embodiment, the increase in macropinocytosis is triggered
by the presence of elevated levels of IdoA2s-GlcNS. In another
preferred embodiment, the increase in macropinocytosis is triggered
by the presence of elevated levels of heparinase I. In another
preferred embodiment, the increase in macropinocytosis is triggered
by the presence of elevated levels of HSPE. In another preferred
embodiment, the increase in macropinocytosis is triggered by
elevated levels of HS2ST1. In another preferred embodiment the
disease is selected from the group consisting of pulmonary
hypertension, interstitial lung disease, acute lung injury (ALI),
acute respiratory distress syndrome (ARDS), sepsis, septic shock,
sarcoidosis of the lung, diabetes, ankylosing spondylitis,
psoriasis, diseases of the endometrium, pulmonary manifestations of
connective tissue diseases, including systemic lupus erythematosus,
rheumatoid arthritis, scleroderma, and polymyositis,
dermatomyositis, bronchiectasis, asbestosis, berylliosis,
silicosis, Histiocytosis X, pneumotitis, smoker's lung,
bronchiolitis obliterans, the prevention of lung scarring due to
tuberculosis and pulmonary fibrosis, other fibrotic diseases such
as myocardial infarction, endomyocardial fibrosis, mediastinal
fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive
massive fibrosis, pneumoconiosis, nephrogenic systemic fibrosis,
keloid, arthrofibrosis, adhesive capsulitis, radiation fibrosis,
fibrocystic breast condition, liver cirrhosis, hepatitis, liver
fibrosis, nonalcoholic fatty liver disease, nonalcoholic
steatohepatitis, sarcoidosis of the lymph nodes, or other organs,
inflammatory bowel disease, Crohn's disease, ulcerative colitis,
primary biliary cirrhosis, pancreatitis, interstitial cystitis,
chronic obstructive pulmonary disease, pneumoconiosis, autoimmune
diseases, angiogenic diseases, wound healing, infections, trauma
injuries and systemic connective tissue diseases including systemic
lupus erythematosus, rheumatoid arthritis, psoriatic arthritis,
juvenile idiopathic arthritis scleroderma, polymyositis, and
dermatomyositis.
[0073] In one embodiment, the present invention discloses a method
of treating a disease comprising increasing macropinocytosis by
administering to a patient suffering from a disease a
therapeutically effective amount of a cell penetrating peptide. In
a preferred embodiment, the peptide is CAR. In another preferred
embodiment, the peptide is tCAR. In yet another preferred
embodiment, the peptide is a variant of CAR with at least 60%
binding affinity to IdoA2s-GlcNS. In still another preferred
embodiment the disease is selected from the group consisting of
pulmonary hypertension, interstitial lung disease, acute lung
injury (ALI), acute respiratory distress syndrome (ARDS), sepsis,
septic shock, sarcoidosis of the lung, diabetes, ankylosing
spondylitis, psoriasis, diseases of the endometrium, pulmonary
manifestations of connective tissue diseases, including systemic
lupus erythematosus, rheumatoid arthritis, scleroderma, and
polymyositis, dermatomyositis, bronchiectasis, asbestosis,
berylliosis, silicosis, Histiocytosis X, pneumotitis, smoker's
lung, bronchiolitis obliterans, the prevention of lung scarring due
to tuberculosis and pulmonary fibrosis, other fibrotic diseases
such as myocardial infarction, endomyocardial fibrosis, mediastinal
fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive
massive fibrosis, pneumoconiosis, nephrogenic systemic fibrosis,
keloid, arthrofibrosis, adhesive capsulitis, radiation fibrosis,
fibrocystic breast condition, liver cirrhosis, hepatitis, liver
fibrosis, nonalcoholic fatty liver disease, nonalcoholic
steatohepatitis, sarcoidosis of the lymph nodes, or other organs,
inflammatory bowel disease, crohn's disease, ulcerative colitis,
primary biliary cirrhosis, pancreatitis, interstitial cystitis,
chronic obstructive pulmonary disease, pneumoconiosis, autoimmune
diseases, angiogenic diseases, wound healing, infections, trauma
injuries and systemic connective tissue diseases including systemic
lupus erythematosus, rheumatoid arthritis, psoriatic arthritis,
juvenile idiopathic arthritis, scleroderma, polymyositis, and
dermatomyositis.
[0074] In one embodiment, the present invention discloses a method
of treating a disease characterized by elevated levels of HPSE, the
method comprising 1) administering a therapeutically effective
amount of a compound that binds to IdoA2s-GlcNS; 2) enhancing
macropinocytosis in the target tissue. In a preferred embodiment,
the disease is selected from the group consisting of pulmonary
hypertension, interstitial lung disease, acute lung injury (ALI),
acute respiratory distress syndrome (ARDS), sepsis, septic shock,
sarcoidosis of the lung, diabetes, ankylosing spondylitis,
psoriasis, diseases of the endometrium, pulmonary manifestations of
connective tissue diseases, including systemic lupus erythematosus,
rheumatoid arthritis, scleroderma, and polymyositis,
dermatomyositis, bronchiectasis, asbestosis, berylliosis,
silicosis, Histiocytosis X, pneumotitis, smoker's lung,
bronchiolitis obliterans, the prevention of lung scarring due to
tuberculosis and pulmonary fibrosis, other fibrotic diseases such
as myocardial infarction, endomyocardial fibrosis, mediastinal
fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive
massive fibrosis, pneumoconiosis, nephrogenic systemic fibrosis,
keloid, arthrofibrosis, adhesive capsulitis, radiation fibrosis,
fibrocystic breast condition, liver cirrhosis, hepatitis, liver
fibrosis, nonalcoholic fatty liver disease, nonalcoholic
steatohepatitis, sarcoidosis of the lymph nodes, or other organs,
inflammatory bowel disease, crohn's disease, ulcerative colitis,
primary biliary cirrhosis, pancreatitis, interstitial cystitis,
chronic obstructive pulmonary disease, pneumoconiosis, autoimmune
diseases, angiogenic diseases, wound healing, infections, trauma
injuries and systemic connective tissue diseases including systemic
lupus erythematosus, rheumatoid arthritis, psoriatic arthritis,
juvenile idiopathic arthritis scleroderma, polymyositis, and
dermatomyositis. In another preferred embodiment, the compound that
binds to IdoA2s-GlcNS is CAR. In another preferred embodiment, the
compound that binds to IdoA2s-GlcNS is tCAR. In yet another
preferred embodiment, the compound that binds to IdoA2s-GlcNS is a
variant of CAR with at least 60% binding affinity to
IdoA2s-GlcNS.
[0075] In one embodiment, the present invention discloses a method
of treating a disease by inhibiting macropinocystosis by
administering a therapeutically effective amount of an irreversible
inhibitor of IdoA2s-GlcNS. In a preferred embodiment, the inhibitor
is rationally designed using current techniques when the target is
known. (http://en.wikipedia.org/wiki/Drug_design) In a preferred
embodiment, the disease is selected from the group consisting of
pulmonary hypertension, interstitial lung disease, acute lung
injury (ALI), acute respiratory distress syndrome (ARDS), sepsis,
septic shock, sarcoidosis of the lung, diabetes, ankylosing
spondylitis, psoriasis, diseases of the endometrium, pulmonary
manifestations of connective tissue diseases, including systemic
lupus erythematosus, rheumatoid arthritis, scleroderma, and
polymyositis, dermatomyositis, bronchiectasis, asbestosis,
berylliosis, silicosis, Histiocytosis X, pneumotitis, smoker's
lung, bronchiolitis obliterans, the prevention of lung scarring due
to tuberculosis and pulmonary fibrosis, other fibrotic diseases
such as myocardial infarction, endomyocardial fibrosis, mediastinal
fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive
massive fibrosis, pneumoconiosis, nephrogenic systemic fibrosis,
keloid, arthrofibrosis, adhesive capsulitis, radiation fibrosis,
fibrocystic breast condition, liver cirrhosis, hepatitis, liver
fibrosis, nonalcoholic fatty liver disease, nonalcoholic
steatohepatitis, sarcoidosis of the lymph nodes, or other organs,
inflammatory bowel disease, crohn's disease, ulcerative colitis,
primary biliary cirrhosis, pancreatitis, interstitial cystitis,
chronic obstructive pulmonary disease, pneumoconiosis, autoimmune
diseases, angiogenic diseases, wound healing, infections, trauma
injuries and systemic connective tissue diseases including systemic
lupus erythematosus, rheumatoid arthritis, psoriatic arthritis,
juvenile idiopathic arthritis scleroderma, polymyositis, and
dermatomyositis.
[0076] In one embodiment, the present invention discloses a method
of treating a disease characterized by elevated levels of HS2ST1,
the method comprising 1) administering a therapeutically effective
amount of a compound that binds to IdoA2s-GlcNS; 2) enhancing
macropinocytosis in the target tissue. In a preferred embodiment,
the disease is selected from the group consisting of pulmonary
hypertension, interstitial lung disease, acute lung injury (ALI),
acute respiratory distress syndrome (ARDS), sepsis, septic shock,
sarcoidosis of the lung, diabetes, ankylosing spondylitis,
psoriasis, diseases of the endometrium, pulmonary manifestations of
connective tissue diseases, including systemic lupus erythematosus,
rheumatoid arthritis, scleroderma, and polymyositis,
dermatomyositis, bronchiectasis, asbestosis, berylliosis,
silicosis, Histiocytosis X, pneumotitis, smoker's lung,
bronchiolitis obliterans, the prevention of lung scarring due to
tuberculosis and pulmonary fibrosis, other fibrotic diseases such
as myocardial infarction, endomyocardial fibrosis, mediastinal
fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive
massive fibrosis, pneumoconiosis, nephrogenic systemic fibrosis,
keloid, arthrofibrosis, adhesive capsulitis, radiation fibrosis,
fibrocystic breast condition, liver cirrhosis, hepatitis, liver
fibrosis, nonalcoholic fatty liver disease, nonalcoholic
steatohepatitis, sarcoidosis of the lymph nodes, or other organs,
inflammatory bowel disease, crohn's disease, ulcerative colitis,
primary biliary cirrhosis, pancreatitis, interstitial cystitis,
chronic obstructive pulmonary disease, pneumoconiosis, autoimmune
diseases, angiogenic diseases, wound healing, infections, trauma
injuries and systemic connective tissue diseases including systemic
lupus erythematosus, rheumatoid arthritis, psoriatic arthritis,
juvenile idiopathic arthritis scleroderma, polymyositis, and
dermatomyositis. In another preferred embodiment, the compound that
binds to IdoA2s-GlcNS is CAR. In another preferred embodiment, the
compound that binds to IdoA2s-GlcNS is tCAR. In yet another
preferred embodiment, the compound that binds to IdoA2s-GlcNS is a
variant of CAR with at least 60% binding affinity to
IdoA2s-GlcNS.
[0077] In one embodiment, the present invention discloses a method
of treating a disease characterized by reduction in
macropinocytosis, the method comprising 1) administering a
therapeutically effective amount of a compound that binds to
IdoA2s-GlcNS; 2) enhancing macropinocytosis in the target tissue.
In a preferred embodiment the disease is selected from the group
consisting of pulmonary hypertension, interstitial lung disease,
acute lung injury (ALI), acute respiratory distress syndrome
(ARDS), sepsis, septic shock, sarcoidosis of the lung, diabetes,
ankylosing spondylitis, psoriasis, diseases of the endometrium,
pulmonary manifestations of connective tissue diseases, including
systemic lupus erythematosus, rheumatoid arthritis, scleroderma,
and polymyositis, dermatomyositis, bronchiectasis, asbestosis,
berylliosis, silicosis, Histiocytosis X, pneumotitis, smoker's
lung, bronchiolitis obliterans, the prevention of lung scarring due
to tuberculosis and pulmonary fibrosis, other fibrotic diseases
such as myocardial infarction, endomyocardial fibrosis, mediastinal
fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive
massive fibrosis, pneumoconiosis, nephrogenic systemic fibrosis,
keloid, arthrofibrosis, adhesive capsulitis, radiation fibrosis,
fibrocystic breast condition, liver cirrhosis, hepatitis, liver
fibrosis, nonalcoholic fatty liver disease, nonalcoholic
steatohepatitis, sarcoidosis of the lymph nodes, or other organs,
inflammatory bowel disease, crohn's disease, ulcerative colitis,
primary biliary cirrhosis, pancreatitis, interstitial cystitis,
chronic obstructive pulmonary disease, pneumoconiosis, autoimmune
diseases, angiogenic diseases, erectile dysfunction, chronic kidney
disease, wound healing, infections, trauma injuries and systemic
connective tissue diseases including systemic lupus erythematosus,
rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic
arthritis scleroderma, polymyositis, and dermatomyositis. In
another preferred embodiment, the substance administered is a
peptide. In another preferred embodiment, the peptide is CAR. In
another preferred embodiment, the peptide is tCAR. In another
preferred embodiment, the peptide is a CAR variant with at least
60% binding affinity to IdoA2s-GlcNS. In yet another preferred
embodiment, the disease is further characterized by elevated levels
of one or more of HPSE, heparinase I and HS2ST1.
[0078] In one embodiment, the present invention discloses a method
of treating a disease characterized by elevated levels of HPSE and
heparinase I comprising the administration of a therapeutically
effective amount of a composition consisting of:
[0079] 1) at least one peptide selected from the group consisting
of CAR, tCAR or a CAR variant with at least 60% binding affinity to
IdoA2s-GlcNS; and
[0080] 2) at least one other therapeutic agent.
In a preferred embodiment, the therapeutic agent is selected from
the group consisting of small molecules, polypeptides, peptides,
peptidomimetics, nucleic acid-molecules, cells and viruses and the
like.
[0081] In one embodiment, the present invention discloses a method
of treating a disease comprising increasing the localized efficacy
of gene therapy by co-administering a therapeutically effective
amount of a cell penetrating peptide and a gene therapy vector. In
a preferred embodiment, the peptide is CAR. In another preferred
embodiment, the peptide is tCAR. In another preferred embodiment,
the peptide is a variant of CAR with at least 60% binding affinity
to IdoA2s-GlcNS.
[0082] In one embodiment, the present invention discloses an
apparatus for determining elevated levels of disease markers
wherein the disease markers are selected from the group consisting
of HPSE, heparinase I, and HS2ST1. In a preferred embodiment, the
apparatus further comprises imaging agents which bind to the
disease markers such that the imaging agents convey the presence
and/or level of the disease marker in the sample.
Examples
[0083] Recent experiments (Urakami et al. 2011; Toba et al. (in
submission)) have demonstrated the ability of the pulmonary
hypertensive homing, cell-penetrating peptide CARSKNKDC (CAR) (FIG.
2) to enhance the effects of co-administered vasodilators (fasudil,
Y-27632, imatimib, and sildenafil) in selectively lowering
pulmonary pressure in animal models of pulmonary arterial
hypertension (PAH). Here we provide a hypothesis for CAR's
mechanism of action and identify a putative receptor.
Methods
[0084] We examined data on enzymatic specificity from the
literature and combined it with genetic expression in a porcine
model of pulmonary hypertension to arrive at a likely target for
CAR. In previous experiments, cell-surface heparan sulfate (HS) was
shown to be necessary for both CAR binding and internalization
(Jarvinen et al. 2007). When treated with heparinase I and III,
binding of CAR to Chinese hamster ovary cells was greatly reduced.
This suggested that CAR's specific binding and internalization is
mediated by the presence of HS moieties on the surface of the
target cell.
[0085] 20-30 kg Yucatan Micropigs underwent surgical anastomosis of
the left pulmonary artery to the descending aorta, resulting in
pulmonary arterial hypertension (Rothman et al. 2011). Endovascular
samples were obtained from 2-3 mm arteries with an endoarterial
biopsy catheter at baseline (prior to surgery), and from the
hypertensive left lung 7, 21, 60 and 180 days after surgery. RNA
was isolated from biopsy samples and loaded into an Affymetrix
GeneChip Porcine Whole Genome Array containing 20, 201 Sus scrofa
genes. Gene expression level differences were analyzed using
GeneSpring, and gene expression fold changes relative to baseline
were calculated (Rothman et al. 2013).
Results/Hypothesis
[0086] In a surgical shunt model of PAH, we found elevated gene
expression levels of heparanase, a substrate specific enzyme
responsible for the cleavage of specific HS moieties (Table 1).
Interestingly, heparinase I and heparanase do not have the same HS
substrate specificity, and one HS moiety in particular,
IdoA2S-GlcNS, is cleaved by heparinase I but not heparanase (Table
2). Our PAH gene expression data also revealed markedly elevated
levels of the heparan sulfate 2-O-sulfotransferase 1 (HS2ST1) and
heparan sulfate 6-O-sulfotransferase 3 (HS6ST2) genes, which encode
the HS2ST1 and HS6ST3 enzymes and are necessary for synthesis of
the HS moieties resistant to heparanase (Table 1).
TABLE-US-00001 TABLE 1 PAH Gene Expression Data Fold Fold Fold Fold
Change Change Change Change Gene Day 7/ Day 21/ Day 60/ Day 180/
Symbol Name Base Base Base Base HPSE Heparanase 1.59 13.24 12.75
2.84 HS2ST1 Heparan 22.40 93.98 26.63 140.44 sulfate 2-O-sulfo-
transferase 1 HS6ST3 Heparan 1.12 21.53 6.33 2.70 sulfate
6-O-sulfo- transferase 3
[0087] Heparanase expression was shown to be upregulated in our PAH
gene expression data. Since CAR has demonstrated selective homing
in multiple models of PAH and it is known that CAR binding and
internalization requires heparan sulfate receptors on target cell
surfaces, CAR is most likely binding to a HS moiety that is
resistant to heparanase. In a recent study, three HS moieties were
shown to be resistant to heparanase (Peterson et al. 2010), but
only one of the three is cleaved by heparinase I (Wei et al. 2005),
an enzyme which blocks CAR internalization (Jarvinen et al. 2007).
Based on this enzyme specificity, we identify IdoA2S-GlcNS as a
putative HS moiety to which CAR binds.
TABLE-US-00002 TABLE 2 Comparison of heparan sulfate substrate
specificity Heparan sulfate moiety Heparinase I cleavage Heparanase
cleavage (below) (EC 4.2.2.7) (EC 3.2.1.166) GlcA-GlcNAc6S No No
GlcA-GlcNS No No IdoA2S-GlcNS Yes No
[0088] One possible mechanism by which CAR could facilitate the
selective uptake of co-administered drugs is through heparan
sulfate-mediated macropinocytosis (FIG. 5). Macropinocytosis is a
non-clathrin, non-caveolin, lipid raft-dependent form of
endocytosis that allows for the regulated internalization of
extracellular solute molecules. Studies have described the role of
heparan sulfate as the receptor for lipid raft-dependent
macropinocytotic internalization (Fan et al. 2007), and
macropinocytosis has also been shown to underly the internalization
of other cationic cell-penetrating peptides (Lim et al. 2011;
Kaplan et al. 2005; Nakase et al. 2004). Heparan sulfate mediated
macropinocytosis could explain CAR's ability to increase the
localized concentration of co-administered drugs without requiring
the drugs to be conjugated to CAR.
[0089] Detailed description of CAR peptide binding and
internalization mechanism of action is shown in FIG. 3.
Specifically, healthy glycocalyx located on endothelial cell
surface facing bloodstream are shown (FIG. 3a). Due to the full,
in-tack glycocalyx layer, CAR cannot access its unique heparan
sulfate receptors. Despite some drug molecules passively diffusing
through the plasma membrane, the majority of drug will not be
internalized into the healthy cell. Upon PAH injury, heparanase
expression levels increase, resulting in selective enzymatic
cleavage of some heparan sulfate chains and modification of the
glycocalyx (FIG. 3b). HS variants resistant to heparanase cleavage
remain in-tact and accessible to CAR, allowing CAR to bind to its
HS receptors. Next is the initiation of heparan sulfate-mediated
macropinocytosis (FIG. 3c). Binding of CAR to its HS receptors
trigger lipid raft formation and membrane ruffling, causing an
inward folding of the plasma membrane and engulfment of surrounding
extracellular components (like CAR and drug molecules). Vesicles
(called macropinosomes) containing CAR and drug molecules are
formed and internalized into the cell (FIG. 3d). Finally, the
reduced intracellular pH causes the macropinosome to dissociate,
releasing its contents (CAR and drug) into the diseased cell (FIG.
3e).
CONCLUSIONS
[0090] These data identify a specific HS moiety (IdoA2S-GlcNS) as a
possible receptor for CAR that could be present in pulmonary
hypertensive tissues, in which most other HS moieties have been
cleaved by high levels of heparanase. We further hypothesize that
heparan sulfate-mediated macropinocytosis could be a plausible
mechanism by which CAR binding could promote the internalization of
co-administered compounds. Experiments are currently underway to
test and refine these hypotheses, while CAR is being developed as a
therapeutic adjuvant for PAH.
Abbreviations
[0091] CAR--a 9 amino acid cyclic peptide CARSKNKDC (where the
amino acids C=cysteine, A=alanine, R=arginine, S=serine, K=lysine,
N=asparagine, D=aspartic acid) TAT--trans-activator of
transcription protein PAH--pulmonary arterial hypertension
GTx--gene therapy EC--enzyme classification HIV--human
immunodeficiency virus MFN2--mitofusion-2 gene HPSE--heparanase
HS--heparan sulfate HS2ST1--heparan sulfate 2-O-sulfotransferase 1
gene RNA--ribonucleic acid mPAP--mean pulmonary arterial pressure
mmHG--millimeters of mercury cDNA--complementary deoxyribonucleic
acid (DNA) MFN2-CAR--mitofusion-2 gene therapy combined with CAR
peptide (co-administration) VSMC--vascular smooth muscle cells
tCAR--truncated CAR (CARSKNK) CAR variants (take from previous
patent apps) RV/LV-S--right ventricle to left ventricle plus septum
(weight ratio of) Sugen Hypoxia Rats--rats that are given sugen (a
vascular endothelial growth factor [VEGF] inhibitor), then placed
in a hypoxia (10% oxygen) chamber CH-SU Rats--chronic hypoxia-sugen
rats SMP--selective macropinocytosis promoter
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Sequence CWU 1
1
219PRTArtificial SequenceSynthesized 1Cys Ala Arg Ser Lys Asn Lys
Asp Cys1 527PRTArtificial SequenceSynthesized 2Cys Ala Arg Ser Lys
Asn Lys1 5
* * * * *
References