U.S. patent application number 14/175319 was filed with the patent office on 2014-07-03 for use of sdf-1 to mitigate scar formation.
This patent application is currently assigned to Juventas Therapeutics, Inc.. The applicant listed for this patent is The Cleveland Clinic Foundation, Juventas Therapeutics, Inc.. Invention is credited to Rahul Aras, Matthew Kiedrowski, Joseph Pastore, Marc S. Penn.
Application Number | 20140186419 14/175319 |
Document ID | / |
Family ID | 40796113 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140186419 |
Kind Code |
A1 |
Penn; Marc S. ; et
al. |
July 3, 2014 |
USE OF SDF-1 TO MITIGATE SCAR FORMATION
Abstract
The subject matter provided herein relates to method for
inhibiting or mitigating scar formation in a wound of the skin, by
increasing the concentration of SDF-1 in, or proximate to, the
wound. As described herein SDF-1 protein or an SDF-1 expression
vector can be administered to a wound or the area proximate a wound
by providing a therapeutically effective amount of SDF-1 protein or
an SDF-1 expression vector.
Inventors: |
Penn; Marc S.; (Beachwood,
OH) ; Kiedrowski; Matthew; (Cleveland, OH) ;
Aras; Rahul; (Broadview Heights, OH) ; Pastore;
Joseph; (Mentor, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Juventas Therapeutics, Inc.
The Cleveland Clinic Foundation |
Cleveland
Cleveland |
OH
OH |
US
US |
|
|
Assignee: |
Juventas Therapeutics, Inc.
Cleveland
OH
The Cleveland Clinic Foundation
Cleveland
OH
|
Family ID: |
40796113 |
Appl. No.: |
14/175319 |
Filed: |
February 7, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13650726 |
Oct 12, 2012 |
8679477 |
|
|
14175319 |
|
|
|
|
12808056 |
Jun 14, 2010 |
|
|
|
PCT/US2008/086820 |
Dec 15, 2008 |
|
|
|
13650726 |
|
|
|
|
61013878 |
Dec 14, 2007 |
|
|
|
Current U.S.
Class: |
424/426 ;
424/400; 424/445; 514/44R |
Current CPC
Class: |
A61K 38/195 20130101;
A61P 17/02 20180101; A61K 38/00 20130101; C07K 14/522 20130101;
A61K 48/00 20130101 |
Class at
Publication: |
424/426 ;
514/44.R; 424/400; 424/445 |
International
Class: |
A61K 38/19 20060101
A61K038/19 |
Claims
1. A method for inhibiting and/or mitigating formation of scar
tissue in a wound of the skin, comprising increasing the
concentration of SDF-1 in, or proximate to, the wound by
administering to said wound and/or an area proximate the wound a
therapeutically effective amount of an SDF-1 expression vector.
2. The method according to claim 1, wherein said wound of the skin
is an acute wound selected from a thermal burn, a chemical burn, a
radiation burn, a burn caused by excess exposure to ultraviolet
radiation, an injury sustained during a medical procedure, an
incision, a trauma-induced injury, a cut or a laceration.
3. The method according to claim 1, wherein the wound of the skin
is a chronic wound selected from a pressure sore, a bedsore, a
wound related to diabetes or poor circulation, or a wound resulting
from dermatitis or acne.
4. The method according to claim 1, comprising administering an
SDF-1 expression vector to said wound and/or an area proximate the
wound.
5. The method according to claim 4, wherein said SDF-1 expression
vector is a viral vector.
6. The method according to claim 4, wherein said SDF-1 expression
vector is a non-viral vector.
7. The method according to claim 6, wherein said non-viral vector
is a DNA plasmid.
8. The method according to claim 1, wherein said SDF-1 expression
vector is administered in the form of a pharmaceutical composition
that comprises an SDF-1 expression vector and a pharmaceutically
acceptable carrier.
9. The method according to claim 1, wherein said pharmaceutical
composition is an injectable formulation.
10. The method according to claim 9, wherein said injectable
formulation is administered by injection directly into the wound or
into an area proximate the wound.
11. The method according to claim 1, wherein said SDF-1 expression
vector is administered in the form of a topical formulation.
12. The method according to claim 1, wherein said SDF-1 expression
vector is administered in or on a substrate, solid support or wound
dressing.
13. The method according to claim 12, wherein said SDF-1 expression
vector is administered in or on a substrate, and the substrate is
in the form of a bioresorbable implant.
14. The method according to claim 1, wherein said SDF-1 expression
vector is administered in or on a wound dressing.
15. The method according to claim 1, wherein said SDF-1 expression
vector is administered to the external surface of the wound.
16. The method according to claim 1, wherein said SDF-1 expression
vector is administered as part of a surgical procedure.
17. The method according to claim 1, wherein said SDF-1 expression
vector is administered within 24 hours of the wound occurring.
18. The method according to claim 1, wherein said SDF-1 expression
vector is administered more than 24 hours after the wound occurred.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/650,726, filed Oct. 12, 2012, now allowed, which is a
division of U.S. application Ser. No. 12/808,056, filed Jun. 14,
2010, now abandoned, which is the U.S. national stage application
of International Application No. PCT/US2008/086820, filed Dec. 15,
2008, which claims priority from U.S. Provisional Application No.
61/013,878, filed Dec. 14, 2007; the subject matter of these
applications is incorporated by reference herein in their
entirety.
TECHNICAL FIELD
[0002] The present invention relates to composition and methods of
promoting wound healing in subject.
BACKGROUND
[0003] Wounds (i.e., lacerations or openings) in mammalian tissue
result in tissue disruption and coagulation of the microvasculature
at the wound face. Repair of such tissue represents an orderly,
controlled cellular response to injury. All soft tissue wounds,
regardless of size heal in a similar manner. Tissue growth and
repair are biologic systems wherein cellular proliferation and
angiogenesis occur in the presence of an oxygen gradient. The
sequential morphological and structural changes which occur during
tissue repair have been characterized in great detail and have in
some instances been quantified (Hunt, T. K., et al., "Coagulation
and macrophage stimulation of angiogenesis and wound healing," in
The Surgical Wound, pp. 1-18, ed. F. Dineen & G. Hildrick-Smith
(Lea & Febiger, Philadelphia: 1981)].
[0004] The cellular morphology consists of three distinct zones.
The central avascular wound space is oxygen deficient, acidotic and
hypercarbic, and has high lactate levels. Adjacent to the wound
space is a gradient zone of local anemia (ischemia) which is
populated by dividing fibroblasts. Behind the leading zone is an
area of active collagen synthesis characterized by mature
fibroblasts and numerous newly-formed capillaries (i.e.,
neovascularization). While this new blood vessel growth
(angiogenesis) is necessary for the healing of wound tissue,
angiogenic agents generally are unable to fulfill the long-felt
need of providing the additional biosynthetic effects of tissue
repair. Despite the need for more rapid healing of wounds (i.e.,
severe burns, surgical incisions, lacerations and other trauma), to
date there has been only limited success in accelerating wound
healing with pharmacological agents.
SUMMARY
[0005] The present invention relates to methods and composition of
treating and/or promoting wound healing in a subject. In the
method, SDF-1 is administered directly to the wound or cells
proximate the wound at an amount effective to promote wound
healing. The wound can include any injury to any portion of the
body of a subject. Examples of wounds that can be treated by the
method include acute conditions or wounds; such as thermal burns,
chemical burns, radiation burns, burns caused by excess exposure to
ultraviolet radiation (e.g., sunburn); damage to bodily tissues,
such as the perineum as a result of labor and childbirth; injuries
sustained during medical procedures, such as episiotomies,
trauma-induced injuries including cuts, incisions, excoriations;
injuries sustained from accidents; post-surgical injuries, as well
as chronic conditions; such as pressure sores, bedsores, conditions
related to diabetes and poor circulation, and all types of acne. In
addition, the wound can include dermatitis, such as impetigo,
intertrigo, folliculitis and eczema, wounds following dental
surgery; periodontal disease; wounds following trauma; and tumor
associated wounds.
[0006] In an aspect of the invention, an amount of SDF-1
administered to the wound or cells proximate the wound can be an
amount effective to promote or accelerate wound closure and wound
healing, mitigate scar formation of and/or around the wound,
inhibit apoptosis of cells surrounding or proximate the wound,
and/or facilitate revascularization of the wounded tissue. The
SDF-1 can be administered to cells proximate the wound that include
SDF-1 receptors that are up-regulated as a result of tissue injury
and/or trauma. In an aspect of the invention, the SDF-1 receptor
can comprise CXCR4 and/or CXCR7, and the SDF-1 can be administered
at an amount effect to increase Akt-phosphorylation of the
cells.
[0007] In another aspect of the invention, the SDF-1 can be
administered by expressing SDF-1 in cells proximate the wound
and/or providing a pharmaceutical composition to the wound which
includes SDF-1. The SDF-1 can be expressed from the cells proximate
the wound by genetically modifying the cells by at least one of a
vector, plasmid DNA, electroporation, and nanoparticles to express
SDF-1.
[0008] The present invention also relates to methods and
composition of inhibiting scar formation during wound healing in a
subject. In the method, SDF-1 is administered directly to the wound
or cells proximate the wound at an amount effective to mitigate
scar formation in and/or around the wound. The wound can include
any injury to any portion of the body of a subject. Examples of
wound that can be treated by the method include acute conditions or
wounds; such as thermal burns, chemical burns, radiation burns,
burns caused by excess exposure to ultraviolet radiation (e.g.,
sunburn); damage to bodily tissues, such as the perineum as a
result of labor and childbirth; injuries sustained during medical
procedures, such as episiotomies, trauma-induced injuries including
cuts, incisions, excoriations; injuries sustained from accidents;
post-surgical injuries, as well as chronic conditions; such as
pressure sores, bedsores, conditions related to diabetes and poor
circulation, and all types of acne. In addition, the wound can
include dermatitis such as impetigo, intertrigo, folliculitis and
eczema, wounds following dental surgery; periodontal disease;
wounds following trauma; and tumor associated wounds.
[0009] In an aspect of the invention, an amount of SDF-1
administered to the wound or cells proximate the wound can be an
amount effective to promote or accelerate wound closure and wound
healing, mitigate scar fibrosis of the tissue of and/or around the
wound, inhibit apoptosis of cells surrounding or proximate the
wound, and/or facilitate revascularization of the wounded tissue.
The SDF-1 can be administered to cells proximate the wound that
include SDF-1 receptors that are up-regulated as a result of tissue
injury and/or trauma. In an aspect of the invention, the SDF-1
receptor can comprise CXCR4 and/or CXCR7, and the SDF-1 can be
administered at an amount effect to increase Akt-phosphorylation of
the cells.
[0010] In another aspect of the invention, the SDF-1 can be
administered by expressing SDF-1 in cells proximate the wound
and/or providing a pharmaceutical composition to the wound which
includes SDF-1. The SDF-1 can be expressed from the cells proximate
the wound by genetically modifying the cells by at least one of a
vector, plasmid DNA, electroporation, and nanoparticles to express
SDF-1.
[0011] The present invention further relates to methods and
composition of promoting or accelerating wound closure in a
subject. In the method, SDF-1 is administered directly to the wound
or cells proximate the wound at an amount effective to promote
wound closure. The wound can include any injury to any portion of
the body of a subject. Examples of wound that can be treated by the
method include acute conditions or wounds; such as thermal burns,
chemical burns, radiation burns, burns caused by excess exposure to
ultraviolet radiation (e.g., sunburn); damage to bodily tissues,
such as the perineum as a result of labor and childbirth; injuries
sustained during medical procedures, such as episiotomies,
trauma-induced injuries including cuts, incisions, excoriations;
injuries sustained from accidents; post-surgical injuries, as well
as chronic conditions; such as pressure sores, bedsores, conditions
related to diabetes and poor circulation, and all types of acne. In
addition, the wound can include dermatitis such as impetigo,
intertrigo, folliculitis and eczema, wounds following dental
surgery; periodontal disease; wounds following trauma; and tumor
associated wounds.
[0012] In an aspect of the invention, an amount of SDF-1
administered to the wound or cells proximate the wound can be an
amount effective to promote or accelerate wound closure and wound
healing, mitigate scar formation of and/or around the wound,
inhibit apoptosis of cells surrounding or proximate the wound,
and/or facilitate revascularization of the wounded tissue. The
SDF-1 can be administered to cells proximate the wound that include
SDF-1 receptors that are up-regulated as a result of tissue injury
and/or trauma. In an aspect of the invention, the SDF-1 receptor
can comprise CXCR4 and/or CXCR7, and the SDF-1 can be administered
at an amount effect to increase Akt-phosphorylation of the
cells.
[0013] In another aspect of the invention, the SDF-1 can be
administered by expressing SDF-1 in cells proximate the wound
and/or providing a pharmaceutical composition to the wound which
includes SDF-1. The SDF-1 can be expressed from the cells proximate
the wound by genetically modifying the cells by at least one of a
vector, plasmid DNA, electroporation, and nanoparticles to express
SDF-1.
[0014] The present invention still further relates to a topical
and/or local formulation for promoting wound healing in subject.
The formulation can include an amount of SDF-1 effective to promote
wound closure and inhibit scarring of the wound when the
formulation is administered to the wound.
[0015] The wound can include any injury to any portion of the body
of a subject. Examples of wound that can be treated by the method
include acute conditions or wounds; such as thermal burns, chemical
burns, radiation burns, burns caused by excess exposure to
ultraviolet radiation (e.g., sunburn); damage to bodily tissues,
such as the perineum as a result of labor and childbirth; injuries
sustained during medical procedures, such as episiotomies,
trauma-induced injuries including cuts, incisions, excoriations;
injuries sustained from accidents; post-surgical injuries, as well
as chronic conditions; such as pressure sores, bedsores, conditions
related to diabetes and poor circulation, and all types of acne. In
addition, the wound can include dermatitis such as impetigo,
intertrigo, folliculitis and eczema, wounds following dental
surgery; periodontal disease; wounds following trauma; and tumor
associated wounds.
[0016] The amount of SDF-1 in the wound can also be an amount
effective to promote or accelerate wound healing, mitigate scar
formation of and/or around the wound, inhibit apoptosis of cells
surrounding or proximate the wound, and/or facilitate
revascularization of the wounded tissue. In an aspect of the
invention, the SDF-1 can be in the form of protein or plasmid that
when administered to a cell proximate the wound promotes expression
of SDF-1 from the cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The foregoing and other features of the present invention
will become apparent to those skilled in the art to which the
present invention relates upon reading the following description
with reference to the accompanying drawings.
[0018] FIG. 1 illustrates photographs showing that SDF-1 releasing
scaffolds accelerate wound healing.
[0019] FIG. 2 illustrates plots showing the % Healing over a period
days for porcine wounds treated with SDF-1 protein scaffold, SDF-1
plasma scaffold, Saline scaffold, and no scaffold.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0020] Unless otherwise defined, all technical terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Commonly
understood definitions of molecular biology terms can be found in,
for example, Rieger et al., Glossary of Genetics: Classical and
Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin,
Genes V, Oxford University Press: New York, 1994.
[0021] Methods involving conventional molecular biology techniques
are described herein. Such techniques are generally known in the
art and are described in detail in methodology treatises, such as
Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed.
Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed.
Ausubel et al., Greene Publishing and Wiley-Interscience, New York,
1992 (with periodic updates). Methods for chemical synthesis of
nucleic acids are discussed, for example, in Beaucage and
Carruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucci et al.,
J. Am. Chem. Soc. 103:3185, 1981. Chemical synthesis of nucleic
acids can be performed, for example, on commercial automated
oligonucleotide synthesizers. Immunological methods (e.g.,
preparation of antigen-specific antibodies, immunoprecipitation,
and immunoblotting) are described, e.g., in Current Protocols in
Immunology, ed. Coligan et al., John Wiley & Sons, New York,
1991; and Methods of Immunological Analysis, ed. Masseyeff et al.,
John Wiley & Sons, New York, 1992. Conventional methods of gene
transfer and gene therapy can also be adapted for use in the
present invention. See, e.g., Gene Therapy: Principles and
Applications, ed. T. Blackenstein, Springer Verlag, 1999; Gene
Therapy Protocols (Methods in Molecular Medicine), ed. P. D.
Robbins, Humana Press, 1997; and Retro-vectors for Human Gene
Therapy, ed. C. P. Hodgson, Springer Verlag, 1996.
[0022] The present invention relates to the treatment of a wound
and/or the promotion of wound healing or wound closure in a
mammalian subject by administering to the wound and/or cells
proximate the wound an amount of SDF-1 effective to promote wound
healing, mitigate cell apoptosis, and/or mitigate or inhibit scar
formation in the wound. The present invention also relates to a
method of inhibiting scar formation and/or fibrosis of a wound or
tissue proximate a wound by administering to the wound and/or cells
or tissue proximate the wound an amount of SDF-1 effective to
promote wound healing, mitigate cell apoptosis, and/or mitigate or
inhibit scar formation in the wound. The present invention further
relates to a topical and/or local formulation for treating a wound
comprising SDF-1 or an agent that upregulates expression of SDF-1
in cells of a wound.
[0023] The wound treated by the method and/or compositions of the
present invention can include any injury to any portion of the body
of a subject (e.g., internal wound or external wound) including:
acute conditions or wounds, such as thermal burns, chemical burns,
radiation burns, burns caused by excess exposure to ultraviolet
radiation (e.g., sunburn); damage to bodily tissues, such as the
perineum as a result of labor and childbirth; injuries sustained
during medical procedures, such as episiotomies; trauma-induced
injuries, such as cuts, incisions, excoriations, injuries sustained
as result of accidents, ulcers, such as pressure ulcers, diabetic
ulcers, plaster ulcers, and decubitus ulcer, post-surgical
injuries. The wound can also include chronic conditions or wounds,
such as pressure sores, bedsores, conditions related to diabetes
and poor circulation, and all types of acne. In addition, the wound
can include dermatitis, such as impetigo, intertrigo, folliculitis
and eczema, wounds following dental surgery; periodontal disease;
tumor associated wounds.
[0024] It will be appreciated that the present application is not
limited to the preceding wounds or injuries and that other wounds
or tissue injuries whether acute and/or chronic can be treated by
the compositions and methods of the present invention.
[0025] As used herein, the term "promoting wound healing" or
"promoting healing of a wound" mean augmenting, improving,
increasing, or inducing closure, healing, or repair of a wound.
[0026] As used herein, the terms "treating" and "treatment" refer
to reduction in severity and/or frequency of symptoms, elimination
of symptoms and/or underlying cause, prevention of the occurrence
of symptoms and/or their underlying cause, and improvement or
remediation of damage. Thus, for example, "treating" of a wound
includes increasing healing at a wound site, promoting wound
closure, and decreasing scarring of the wound.
[0027] Mammalian subjects, which will be treated by methods and
compositions of the present invention, can include any mammal, such
as human beings, rats, mice, cats, dogs, goats, sheep, horses,
monkeys, apes, rabbits, cattle, etc. The mammalian subject can be
in any stage of development including adults, young animals, and
neonates. Mammalian subjects can also include those in a fetal
stage of development.
[0028] In accordance with an aspect of the invention, the SDF-1 can
be administered to cells proximate the wound to mitigate apoptosis
of the cells and promote wound healing, promote wound closure,
and/or mitigate scar formation of and/or around the wound. The
cells include cells that express SDF-1 receptors, which are
upregulated as a result of trauma and/or tissue injury. The
up-regulated SDF-1 receptors can include, for example, CXCR4 and/or
CXCR7. It was found that sustained localized administration of
SDF-1 to cells with up-regulated SDF-1 receptors as a result of
tissue injury increases Akt phosphorylation in the cells which in
turn can mitigate apoptosis of the cells. Additionally, long-term
localized administration of SDF-1 to tissue facilitates recruitment
of stem cells and/or progenitor cells, such as endothelial
progenitor cells, expressing CXCR4 and/or CXCR7 to the site of the
wound being treated, which can facilitate revascularization of the
tissue surrounding and/or proximate the wound.
[0029] In one example, the period of time that the SDF-1 is
administered to the cells of the wound and/or proximate the wound
can comprise from about onset of the wound and/or tissue injury to
about days, weeks, or months after tissue injury. It was found that
topical and/or local SDF-1 delivery by protein or plasmid to wounds
was sufficient to increase the rate of healing and wound closure.
Moreover, the SDF-1 treated wounds tended to have less fibrosis
than non-SDF-1 treated wounds, which suggests SDF-1 can mitigate
scarring in treated wounds. It was also found that immediately
after onset of tissue injury, cells in the wound tissue or about
the periphery or the border of the wound up regulate expression of
SDF-1. After about 24 hours, SDF-1 expression by the cells is
reduced. The SDF-1 can be administered after the SDF-1 is reduced
to mitigate apoptosis of the cells.
[0030] SDF-1 in accordance with the present invention can have an
amino acid sequence that is substantially similar to a native
mammalian SDF-1 amino acid sequence. The amino acid sequence of a
number of different mammalian SDF-1 protein are known including
human, mouse, and rat. The human and rat SDF-1 amino acid sequences
are about 92% identical. SDF-1 can comprise two isoforms, SDF-1
alpha and SDF-1 beta, both of which are referred to herein as SDF-1
unless identified otherwise.
[0031] The SDF-1 can have an amino acid sequence substantially
identical to SEQ ID NO: 1. The SDF-1 that is over-expressed can
also have an amino acid sequence substantially similar to one of
the foregoing mammalian SDF-1 proteins. For example, the SDF-1 that
is over-expressed can have an amino acid sequence substantially
similar to SEQ ID NO: 2. SEQ ID NO: 2, which substantially
comprises SEQ ID NO: 1, is the amino acid sequence for human SDF-1
and is identified by GenBank Accession No. NP954637. The SDF-1 that
is over-expressed can also have an amino acid sequence that is
substantially identical to SEQ ID NO: 3. SEQ ID NO: 3 includes the
amino acid sequences for rat SDF and is identified by GenBank
Accession No. AAF01066.
[0032] The SDF-1 in accordance with the present invention can also
be a variant of mammalian SDF-1, such as a fragment, analog and
derivative of mammalian SDF-1. Such variants include, for example,
a polypeptide encoded by a naturally occurring allelic variant of
native SDF-1 gene (i.e., a naturally occurring nucleic acid that
encodes a naturally occurring mammalian SDF-1 polypeptide), a
polypeptide encoded by an alternative splice form of a native SDF-1
gene, a polypeptide encoded by a homolog or ortholog of a native
SDF-1 gene, and a polypeptide encoded by a non-naturally occurring
variant of a native SDF-1 gene.
[0033] SDF-1 variants have a peptide sequence that differs from a
native SDF-1 polypeptide in one or more amino acids. The peptide
sequence of such variants can feature a deletion, addition, or
substitution of one or more amino acids of a SDF-1 variant. Amino
acid insertions are preferably of about 1 to 4 contiguous amino
acids, and deletions are preferably of about 1 to 10 contiguous
amino acids. Variant SDF-1 polypeptides substantially maintain a
native SDF-1 functional activity. Examples of SDF-1 polypeptide
variants can be made by expressing nucleic acid molecules within
the invention that feature silent or conservative changes. One
example of an SDF-1 variant is listed in U.S. Pat. No. 7,405,195,
which is herein incorporated by reference in its entirety.
[0034] SDF-1 polypeptide fragments corresponding to one or more
particular motifs and/or domains or to arbitrary sizes, are within
the scope of the present invention. Isolated peptidyl portions of
SDF-1 can be obtained by screening peptides recombinantly produced
from the corresponding fragment of the nucleic acid encoding such
peptides. For example, an SDF-1 polypeptides of the present
invention may be arbitrarily divided into fragments of desired
length with no overlap of the fragments, or preferably divided into
overlapping fragments of a desired length. The fragments can be
produced recombinantly and tested to identify those peptidyl
fragments, which can function as agonists of native CXCR-4
polypeptides.
[0035] Variants of SDF-1 polypeptides can also include recombinant
forms of the SDF-1 polypeptides. Recombinant polypeptides preferred
by the present invention, in addition to SDF-1 polypeptides, are
encoded by a nucleic acid that can have at least 70% sequence
identity with the nucleic acid sequence of a gene encoding a
mammalian SDF-1.
[0036] SDF-1 variants can include agonistic forms of the protein
that constitutively express the functional activities of native
SDF-1. Other SDF-1 variants can include those that are resistant to
proteolytic cleavage, as for example, due to mutations, which alter
protease target sequences. Whether a change in the amino acid
sequence of a peptide results in a variant having one or more
functional activities of a native SDF-1 can be readily determined
by testing the variant for a native SDF-1 functional activity.
[0037] The SDF-1 nucleic acid that encodes the SDF-1 protein can be
a native or non-native nucleic acid and be in the form of RNA or in
the form of DNA (e.g., cDNA, genomic DNA, and synthetic DNA). The
DNA can be double-stranded or single-stranded, and if
single-stranded may be the coding (sense) strand or non-coding
(anti-sense) strand. The nucleic acid coding sequence that encodes
SDF-1 may be substantially similar to a nucleotide sequence of the
SDF-1 gene, such as nucleotide sequence shown in SEQ ID NO: 4 and
SEQ ID NO: 5. SEQ ID NO: 4 and SEQ ID NO: 5 comprise, respectively,
the nucleic acid sequences for human SDF-1 and rat SDF-1 and are
substantially similar to the nucleic sequences of GenBank Accession
No. NM199168 and GenBank Accession No. AF189724. The nucleic acid
coding sequence for SDF-1 can also be a different coding sequence
which, as a result of the redundancy or degeneracy of the genetic
code, encodes the same polypeptide as SEQ ID NO: 1, SEQ ID NO: 2,
and SEQ ID NO: 3.
[0038] Other nucleic acid molecules that encode SDF-1 within the
invention are variants of a native SDF-1, such as those that encode
fragments, analogs and derivatives of native SDF-1. Such variants
may be, for example, a naturally occurring allelic variant of a
native SDF-1 gene, a homolog or ortholog of a native SDF-1 gene, or
a non-naturally occurring variant of a native SDF-1 gene. These
variants have a nucleotide sequence that differs from a native
SDF-1 gene in one or more bases. For example, the nucleotide
sequence of such variants can feature a deletion, addition, or
substitution of one or more nucleotides of a native SDF-1 gene.
Nucleic acid insertions are preferably of about 1 to 10 contiguous
nucleotides, and deletions are preferably of about 1 to 10
contiguous nucleotides.
[0039] In other applications, variant SDF-1 displaying substantial
changes in structure can be generated by making nucleotide
substitutions that cause less than conservative changes in the
encoded polypeptide. Examples of such nucleotide substitutions are
those that cause changes in (a) the structure of the polypeptide
backbone; (b) the charge or hydrophobicity of the polypeptide; or
(c) the bulk of an amino acid side chain. Nucleotide substitutions
generally expected to produce the greatest changes in protein
properties are those that cause non-conservative changes in codons.
Examples of codon changes that are likely to cause major changes in
protein structure are those that cause substitution of (a) a
hydrophilic residue (e.g., serine or threonine), for (or by) a
hydrophobic residue (e.g., leucine, isoleucine, phenylalanine,
valine or alanine); (b) a cysteine or proline for (or by) any other
residue; (c) a residue having an electropositive side chain (e.g.,
lysine, arginine, or histidine), for (or by) an electronegative
residue (e.g., glutamine or aspartine); or (d) a residue having a
bulky side chain (e.g., phenylalanine), for (or by) one not having
a side chain, (e.g., glycine).
[0040] Naturally occurring allelic variants of a native SDF-1 gene
within the invention are nucleic acids isolated from mammalian
tissue that have at least 70% sequence identity with a native SDF-1
gene, and encode polypeptides having structural similarity to a
native SDF-1 polypeptide. Homologs of a native SDF-1 gene within
the invention are nucleic acids isolated from other species that
have at least 70% sequence identity with the native gene, and
encode polypeptides having structural similarity to a native SDF-1
polypeptide. Public and/or proprietary nucleic acid databases can
be searched to identify other nucleic acid molecules having a high
percent (e.g., 70% or more) sequence identity to a native SDF-1
gene.
[0041] Non-naturally occurring SDF-1 gene variants are nucleic
acids that do not occur in nature (e.g., are made by the hand of
man), have at least 70% sequence identity with a native SDF-1 gene,
and encode polypeptides having structural similarity to a native
SDF-1 polypeptide. Examples of non-naturally occurring SDF-1 gene
variants are those that encode a fragment of a native SDF-1
protein, those that hybridize to a native SDF-1 gene or a
complement of to a native SDF-1 gene under stringent conditions,
and those that share at least 65% sequence identity with a native
SDF-1 gene or a complement of a native SDF-1 gene.
[0042] Nucleic acids encoding fragments of a native SDF-1 gene
within the invention are those that encode, amino acid residues of
native SDF-1. Shorter oligonucleotides that encode or hybridize
with nucleic acids that encode fragments of native SDF-1 can be
used as probes, primers, or antisense molecules. Longer
polynucleotides that encode or hybridize with nucleic acids that
encode fragments of a native SDF-1 can also be used in various
aspects of the invention. Nucleic acids encoding fragments of a
native SDF-1 can be made by enzymatic digestion (e.g., using a
restriction enzyme) or chemical degradation of the full-length
native SDF-1 gene or variants thereof.
[0043] Nucleic acids that hybridize under stringent conditions to
one of the foregoing nucleic acids can also be used in the
invention. For example, such nucleic acids can be those that
hybridize to one of the foregoing nucleic acids under low
stringency conditions, moderate stringency conditions, or high
stringency conditions are within the invention.
[0044] Nucleic acid molecules encoding a SDF-1 fusion protein may
also be used in the invention. Such nucleic acids can be made by
preparing a construct (e.g., an expression vector) that expresses a
SDF-1 fusion protein when introduced into a suitable target cell.
For example, such a construct can be made by ligating a first
polynucleotide encoding a SDF-1 protein fused in frame with a
second polynucleotide encoding another protein such that expression
of the construct in a suitable expression system yields a fusion
protein.
[0045] The nucleic acids encoding SDF-1 can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, hybridization, etc. The nucleic
acids within the invention may additionally include other appended
groups such as peptides (e.g., for targeting target cell receptors
in vivo), or agents facilitating transport across the cell
membrane, hybridization-triggered cleavage. To this end, the
nucleic acids may be conjugated to another molecule, (e.g., a
peptide), hybridization triggered cross-linking agent, transport
agent, hybridization-triggered cleavage agent, etc.
[0046] The SDF-1 can be administered directly to the wound, about
the periphery of the wound or to cells proximate, the wound in
order to mitigate apoptosis of cells proximate the wound and
facilitate angiogenesis to the wounded area as well as accelerate
wound closure and inhibit scarring of the wound. The SDF-1 can be
delivered to the wound or cells proximate the wound by
administering an SDF-1 protein to the wound or cells, or by
introducing an agent into target cells that causes, increases,
and/or upregulates expression of SDF-1 (i.e., SDF-1 agent). The
SDF-1 protein expressed in the target cells can be an expression
product of a genetically modified cell. The target cells can
include cells within or about the periphery of the wound or ex vivo
cells that are biocompatible with tissue being treated. The
biocompatible cells can also include autologous cells that are
harvested from the subject being treated and/or biocompatible
allogeneic or syngeneic cells, such as autologous, allogeneic, or
syngeneic stem cells (e.g., mesenchymal stem cells), progenitor
cells (e.g., multipotent adult progenitor cells) and/or other cells
that are further differentiated and are biocompatible with the
tissue being treated. The cells can include cells that are provided
in skin grafts, bone grafts, engineered tissue, and other tissue
replacement therapies that are used to treat wounds.
[0047] The agent can comprise natural or synthetic nucleic acids,
according to present invention and described above, that are
incorporated into recombinant nucleic acid constructs, typically
DNA constructs, capable of introduction into and replication in the
cell. Such a construct can include a replication system and
sequences that are capable of transcription and translation of a
polypeptide-encoding sequence in a given target cell.
[0048] Other agents can also be introduced into the cells to
promote expression of SDF-1 from the cells. For example, agents
that increase the transcription of a gene encoding SDF-1, increase
the translation of an mRNA encoding SDF-1, and/or those that
decrease the degradation of an mRNA encoding SDF-1 could be used to
increase SDF-1 protein levels. Increasing the rate of transcription
from a gene within a cell can be accomplished by introducing an
exogenous promoter upstream of the gene encoding SDF-1. Enhancer
elements, which facilitate expression of a heterologous gene, may
also be employed.
[0049] Other agents can further include other proteins, chemokines,
and cytokines, that when administered to the target cells can
upregulate expression SDF-1 form the target cells. Such agents can
include, for example: insulin-like growth factor (IGF)-1, which was
shown to upregulate expression of SDF-1 when administered to
mesenchymal stem cells (MSCs) (Circ. Res. 2008, November 21;
103(11):1300-98); sonic hedgehog (Shh), which was shown to
upregulate expression of SDF-1 when administered to adult
fibroblasts (Nature Medicine, Volume 11, Number 11, November 23);
transforming growth factor .beta. (TGF-.beta.); which was shown to
upregulate expression of SDF-1 when administered to human
peritoneal mesothelial cells (HPMCs); IL-1.beta., PDG-BF, VEGF,
TNF-.alpha., and PTH, which are shown to upregulate expression of
SDF-1, when administered to primary human osteoblasts (HOBS) mixed
marrow stromal cells (BMSCs), and human osteoblast-like cell lines
(Bone, 2006, April; 38(4): 497-508); thymosin .beta.4, which was
shown to upregulate expression when administered to bone marrow
cells (BMCs) (Curr. Pharm. Des. 2007; 13(31):3245-51; and hypoxia
inducible factor 1.alpha. (HIF-1), which was shown to upregulate
expression of SDF-1 when administered to bone marrow derived
progenitor cells (Cardiovasc. Res. 2008, E. Pub.). These agents can
be used to treat specific wounds or injuries where such cells
capable of upregulating expression of SDF-1 with respect to the
specific cytokine are present or administered.
[0050] One method of introducing the agent into a target cell
involves using gene therapy. Gene therapy in accordance with the
present invention can be used to express SDF-1 protein from a
target cell in vivo or in vitro.
[0051] In an aspect of the invention, the gene therapy can use a
vector including a nucleotide encoding an SDF-1 protein. A "vector"
(sometimes referred to as gene delivery or gene transfer "vehicle")
refers to a macromolecule or complex of molecules comprising a
polynucleotide to be delivered to a target cell, either in vitro or
in vivo. The polynucleotide to be delivered may comprise a coding
sequence of interest in gene therapy. Vectors include, for example,
viral vectors (such as adenoviruses (`Ad`), adeno-associated
viruses (AAV), and retroviruses), liposomes and other
lipid-containing complexes, and other macromolecular complexes
capable of mediating delivery of a polynucleotide to a target
cell.
[0052] Vectors can also comprise other components or
functionalities that further modulate gene delivery and/or gene
expression, or that otherwise provide beneficial properties to the
targeted cells. Such other components include, for example,
components that influence binding or targeting to cells (including
components that mediate cell-type or tissue-specific binding);
components that influence uptake of the vector nucleic acid by the
cell; components that influence localization of the polynucleotide
within the cell after uptake (such as agents mediating nuclear
localization); and components that influence expression of the
polynucleotide. Such components also might include markers, such as
detectable and/or selectable markers that can be used to detect or
select for cells that have taken up and are expressing the nucleic
acid delivered by the vector. Such components can be provided as a
natural feature of the vector (such as the use of certain viral
vectors which have components or functionalities mediating binding
and uptake), or vectors can be modified to provide such
functionalities.
[0053] Selectable markers can be positive, negative or
bifunctional. Positive selectable markers allow selection for cells
carrying the marker, whereas negative selectable markers allow
cells carrying the marker to be selectively eliminated. A variety
of such marker genes have been described, including bifunctional
(i.e. positive/negative) markers (see, e.g., Lupton, S., WO
92/08796, published May 29, 1992; and Lupton, S., WO 94/28143,
published Dec. 8, 1994). Such marker genes can provide an added
measure of control that can be advantageous in gene therapy
contexts. A large variety of such vectors are known in the art and
are generally available.
[0054] Vectors for use in the present invention include viral
vectors, lipid based vectors and other non-viral vectors that are
capable of delivering a nucleotide according to the present
invention to the target cells. The vector can be a targeted vector,
especially a targeted vector that preferentially binds to cells of
proximate the wound. Viral vectors for use in the invention can
include those that exhibit low toxicity to a target cell and induce
production of therapeutically useful quantities of SDF-1 protein in
a tissue-specific manner.
[0055] Examples of viral vectors are those derived from adenovirus
(Ad) or adeno-associated virus (AAV). Both human and non-human
viral vectors can be used and the recombinant viral vector can be
replication-defective in humans. Where the vector is an adenovirus,
the vector can comprise a polynucleotide having a promoter operably
linked to a gene encoding the SDF-1 protein and is
replication-defective in humans.
[0056] Other viral vectors that can be use in accordance with the
present invention include herpes simplex virus (HSV)-based vectors.
HSV vectors deleted of one or more immediate early genes (IE) are
advantageous because they are generally non-cytotoxic, persist in a
state similar to latency in the target cell, and afford efficient
target cell transduction. Recombinant HSV vectors can incorporate
approximately 30 kb of heterologous nucleic acid.
[0057] Retroviruses, such as C-type retroviruses and lentiviruses,
might also be used in the invention. For example, retroviral
vectors may be based on murine leukemia virus (MLV). See, e.g., Hu
and Pathak, Pharmacol. Rev. 52:493-511, 2000 and Fong et al., Crit.
Rev. Ther. Drug Carrier Syst. 17:1-60, 2000. MLV-based vectors may
contain up to 8 kb of heterologous (therapeutic) DNA in place of
the viral genes. The heterologous DNA may include a tissue-specific
promoter and an SDF-1 nucleic acid. In methods of delivery to cells
proximate the wound, it may also encode a ligand to a tissue
specific receptor.
[0058] Additional retroviral vectors that might be used are
replication-defective lentivirus-based vectors, including human
immunodeficiency (HIV)-based vectors. See, e.g., Vigna and Naldini,
J. Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Virol.
72:8150-8157, 1998. Lentiviral vectors are advantageous in that
they are capable of infecting both actively dividing and
non-dividing cells. They are also highly efficient at transducing
human epithelial cells.
[0059] Lentiviral vectors for use in the invention may be derived
from human and non-human (including SIV) lentiviruses. Examples of
lentiviral vectors include nucleic acid sequences required for
vector propagation as well as a tissue-specific promoter operably
linked to a SDF-1 gene. These former may include the viral LTRs, a
primer binding site, a polypurine tract, att sites, and an
encapsidation site.
[0060] A lentiviral vector may be packaged into any suitable
lentiviral capsid. The substitution of one particle protein with
another from a different virus is referred to as "pseudotyping".
The vector capsid may contain viral envelope proteins from other
viruses, including murine leukemia virus (MLV) or vesicular
stomatitis virus (VSV). The use of the VSV G-protein yields a high
vector titer and results in greater stability of the vector virus
particles.
[0061] Alphavirus-based vectors, such as those made from semliki
forest virus (SFV) and sindbis virus (SIN), might also be used in
the invention. Use of alphaviruses is described in Lundstrom, K.,
Intervirology 43:247-257, 2000 and Perri et al., Journal of
Virology 74:9802-9807, 2000.
[0062] Recombinant, replication-defective alphavirus vectors are
advantageous because they are capable of high-level heterologous
(therapeutic) gene expression, and can infect a wide target cell
range. Alphavirus replicons may be targeted to specific cell types
by displaying on their virion surface a functional heterologous
ligand or binding domain that would allow selective binding to
target cells expressing a cognate binding partner. Alphavirus
replicons may establish latency, and therefore long-term
heterologous nucleic acid expression in a target cell. The
replicons may also exhibit transient heterologous nucleic acid
expression in the target cell.
[0063] In many of the viral vectors compatible with methods of the
invention, more than one promoter can be included in the vector to
allow more than one heterologous gene to be expressed by the
vector. Further, the vector can comprise a sequence which encodes a
signal peptide or other moiety which facilitates the secretion of a
SDF-1 gene product from the target cell.
[0064] To combine advantageous properties of two viral vector
systems, hybrid viral vectors may be used to deliver a SDF-1
nucleic acid to a target tissue. Standard techniques for the
construction of hybrid vectors are well-known to those skilled in
the art. Such techniques can be found, for example, in Sambrook, et
al., In Molecular Cloning: A laboratory manual. Cold Spring Harbor,
N.Y. or any number of laboratory manuals that discuss recombinant
DNA technology. Double-stranded AAV genomes in adenoviral capsids
containing a combination of AAV and adenoviral ITRs may be used to
transduce cells. In another variation, an AAV vector may be placed
into a "gutless", "helper-dependent" or "high-capacity" adenoviral
vector. Adenovirus/AAV hybrid vectors are discussed in Lieber et
al., J. Virol. 73:9314-9324, 1999. Retrovirus/adenovirus hybrid
vectors are discussed in Zheng et al., Nature Biotechnol.
18:176-186, 2000. Retroviral genomes contained within an adenovirus
may integrate within the target cell genome and effect stable SDF-1
gene expression.
[0065] Other nucleotide sequence elements which facilitate
expression of the SDF-1 gene and cloning of the vector are further
contemplated. For example, the presence of enhancers upstream of
the promoter or terminators downstream of the coding region, for
example, can facilitate expression.
[0066] In accordance with another aspect of the present invention,
a tissue-specific promoter, can be fused to a SDF-1 gene. By fusing
such tissue specific promoter within the adenoviral construct,
transgene expression is limited to a particular tissue. The
efficacy of gene expression and degree of specificity provided by
tissue specific promoters can be determined, using the recombinant
adenoviral system of the present invention.
[0067] In addition to viral vector-based methods, non-viral methods
may also be used to introduce a SDF-1 nucleic acid into a target
cell. A review of non-viral methods of gene delivery is provided in
Nishikawa and Huang, Human Gene Ther. 12:861-870, 2001. An example
of a non-viral gene delivery method according to the invention
employs plasmid DNA to introduce a SDF-1 nucleic acid into a cell.
Plasmid-based gene delivery methods are generally known in the
art.
[0068] Synthetic gene transfer molecules can be designed to form
multimolecular aggregates with plasmid DNA. These aggregates can be
designed to bind to a target cell. Cationic amphiphiles, including
lipopolyamines and cationic lipids, may be used to provide
receptor-independent SDF-1 nucleic acid transfer into target cells
(e.g., cardiomyocytes). In addition, preformed cationic liposomes
or cationic lipids may be mixed with plasmid DNA to generate
cell-transfecting complexes. Methods involving cationic lipid
formulations are reviewed in Feigner et al., Ann N.Y. Acad. Sci.
772:126-139, 1995 and Lasic and Templeton, Adv. Drug Delivery Rev.
20:221-266, 1996. For gene delivery, DNA may also be coupled to an
amphipathic cationic peptide (Fominaya et al., J. Gene Med.
2:455-464, 2000).
[0069] Methods that involve both viral and non-viral based
components may be used according to the invention. For example, an
Epstein Barr virus (EBV)-based plasmid for therapeutic gene
delivery is described in Cui et al., Gene Therapy 8:1508-1513,
2001. Additionally, a method involving a DNA/ligand/polycationic
adjunct coupled to an adenovirus is described in Curiel, D. T.,
Nat. Immun. 13:141-164, 1994.
[0070] Additionally, the SDF-1 nucleic acid can be introduced into
the target cell by transfecting the target cells using
electroporation techniques. Electroporation techniques are well
known and can be used to facilitate transfection of cells using
plasmid DNA.
[0071] Vectors that encode the expression of SDF-1 can be delivered
to the target cell in the form of an injectable preparation
containing pharmaceutically acceptable carrier, such as saline, as
necessary. Other pharmaceutical carriers, formulations and dosages
can also be used in accordance with the present invention.
[0072] Where the target cell comprises a cell proximate the wound
being treated, the vector can be delivered by direct injection at
an amount sufficient for the SDF-1 protein to be expressed to a
degree which allows for highly effective therapy. By injecting the
vector directly into or about the periphery of the wound, it is
possible to target the vector transfection rather effectively, and
to minimize loss of the recombinant vectors. This type of injection
enables local transfection of a desired number of cells, especially
about the wound, thereby maximizing therapeutic efficacy of gene
transfer, and minimizing the possibility of an inflammatory
response to viral proteins.
[0073] Where the target cell is a cultured cell that is later
transplanted into wound (e.g., tissue graft), the vectors can be
delivered by direct injection into the culture medium. A SDF-1
nucleic acid transfected into cells may be operably linked to a
regulatory sequence.
[0074] The transfected target cells can then be transplanted to the
wound by well known transplantation techniques, such as graft
transplantation. By first transfecting the target cells in vitro
and then transplanting the transfected target cells to the wound,
the possibility of inflammatory response in the tissue proximate
the wound is minimized compared to direct injection of the vector
into cells proximate the wound.
[0075] SDF-1 can be expressed for any suitable length of time
within the target cell, including transient expression and stable,
long-term expression. In one aspect of the invention, the SDF-1
nucleic acid will be expressed in therapeutic amounts for a defined
length of time effective to mitigate apoptosis in the cells
proximate the wound and/or to promote stem cell or progenitor cell
homing to the wound. This amount of time can be that amount effect
to promote healing of the wound, accelerate closure of the wound,
and/or inhibit scar formation.
[0076] A therapeutic amount is an amount, which is capable of
producing a medically desirable result in a treated animal or
human. As is well known in the medical arts, dosage for any one
animal or human depends on many factors, including the subject's
size, body surface area, age, the particular composition to be
administered, sex, time and route of administration, general
health, and other drugs being administered concurrently. Specific
dosages of proteins and nucleic acids can be determined readily
determined by one skilled in the art using the experimental methods
described below.
[0077] The SDF-1 protein or agent, which causes, increases, and/or
upregulates expression of SDF-1 from target cells, can be
administered to the cells of the wound, cells proximate wound, or
cells administered to the wound (e.g., MSCs transfected to express
SDF-1) neat or in a pharmaceutical composition. The pharmaceutical
composition can provide localized release of the SDF-1 or agent to
the cells proximate the wound, cells being treated, or cells
administered to the wound. Pharmaceutical compositions in
accordance with the invention will generally include an amount of
SDF-1 or agent admixed with an acceptable pharmaceutical diluent or
excipient, such as a sterile aqueous solution, to give a range of
final concentrations, depending on the intended use. The techniques
of preparation are generally well known in the art as exemplified
by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing
Company, 1980, incorporated herein by reference. Moreover, for
human administration, preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biological Standards.
[0078] The pharmaceutical composition can be in a unit dosage
injectable form (e.g., solution, suspension, and/or emulsion).
Examples of pharmaceutical formulations that can be used for
injection include sterile aqueous solutions or dispersions and
sterile powders for reconstitution into sterile injectable
solutions or dispersions. The carrier can be a solvent or
dispersing medium containing, for example, water, ethanol, polyol
(e.g., glycerol, propylene glycol, liquid polyethylene glycol, and
the like), suitable mixtures thereof and vegetable oils.
[0079] Proper fluidity can be maintained, for example, by the use
of a coating, such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil,
olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and
esters, such as isopropyl myristate, may also be used as solvent
systems for compound compositions
[0080] Additionally, various additives which enhance the stability,
sterility, and isotonicity of the compositions, including
antimicrobial preservatives, antioxidants, chelating agents, and
buffers, can be added. Prevention of the action of microorganisms
can be ensured by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, and the
like. In many cases, it will be desirable to include isotonic
agents, for example, sugars, sodium chloride, and the like.
Prolonged absorption of the injectable pharmaceutical form can be
brought about by the use of agents delaying absorption, for
example, aluminum monostearate and gelatin. According to the
present invention, however, any vehicle, diluent, or additive used
would have to be compatible with the compounds.
[0081] Sterile injectable solutions can be prepared by
incorporating the compounds utilized in practicing the present
invention in the required amount of the appropriate solvent with
various amounts of the other ingredients, as desired.
[0082] Pharmaceutical "slow release" capsules or "sustained
release" compositions or preparations may be used and are generally
applicable. Slow release formulations are generally designed to
give a constant drug level over an extended period and may be used
to deliver the SDF-1 or agent. The slow release formulations are
typically implanted in the vicinity of the wound site, for example,
at the site of cell expressing CXCR4 and/or CXCR7 in or about the
wound.
[0083] Examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
SDF-1 or agent, which matrices are in the form of shaped articles,
e.g., films or microcapsule. Examples of sustained-release matrices
include polyesters; hydrogels, for example,
poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol);
polylactides, e.g., U.S. Pat. No. 3,773,919; copolymers of
L-glutamic acid and .gamma. ethyl-L-glutamate; non-degradable
ethylene-vinyl acetate; degradable lactic acid-glycolic acid
copolymers, such as the LUPRON DEPOT (injectable microspheres
composed of lactic acid-glycolic acid copolymer and leuprolide
acetate); and poly-D-(-)-3-hydroxybutyric acid.
[0084] While polymers, such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated, SDF-1 or the agent can remain in the body for a long
time, and may denature or aggregate as a result of exposure to
moisture at 37.degree. C., thus reducing biological activity and/or
changing immunogenicity. Rational strategies are available for
stabilization depending on the mechanism involved. For example, if
the aggregation mechanism involves intermolecular S--S bond
formation through thio-disulfide interchange, stabilization is
achieved by modifying sulfhydryl residues, lyophilizing from acidic
solutions, controlling moisture content, using appropriate
additives, developing specific polymer matrix compositions, and the
like.
[0085] In certain embodiments, liposomes and/or nanoparticles may
also be employed with the SDF-1 or agent. The formation and use of
liposomes is generally known to those of skill in the art, as
summarized below.
[0086] Liposomes are formed from phospholipids that are dispersed
in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs)). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 .ANG.,
containing an aqueous solution in the core.
[0087] Phospholipids can form a variety of structures other than
liposomes when dispersed in water, depending on the molar ratio of
lipid to water. At low ratios, the liposome is the preferred
structure. The physical characteristics of liposomes depend on pH,
ionic strength and the presence of divalent cations. Liposomes can
show low permeability to ionic and polar substances, but at
elevated temperatures undergo a phase transition which markedly
alters their permeability. The phase transition involves a change
from a closely packed, ordered structure, known as the gel state,
to a loosely packed, less-ordered structure, known as the fluid
state. This occurs at a characteristic phase-transition temperature
and results in an increase in permeability to ions, sugars and
drugs.
[0088] Liposomes interact with cells via four different mechanisms:
Endocytosis by phagocytic cells of the reticuloendothelial system
such as macrophages and neutrophils; adsorption to the cell
surface, either by nonspecific weak hydrophobic or electrostatic
forces, or by specific interactions with cell-surface components;
fusion with the plasma cell membrane by insertion of the lipid
bilayer of the liposome into the plasma membrane, with simultaneous
release of liposomal contents into the cytoplasm; and by transfer
of liposomal lipids to cellular or subcellular membranes, or vice
versa, without any association of the liposome contents. Varying
the liposome formulation can alter which mechanism is operative,
although more than one may operate at the same time.
[0089] Nanocapsules can generally entrap compounds in a stable and
reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) should be designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use in the present
invention, and such particles may be are easily made.
[0090] For preparing pharmaceutical compositions from the compounds
of the present invention, pharmaceutically acceptable carriers can
be in any suitable form (e.g., solids, liquids, gels, etc.). A
solid carrier can be one or more substances which may also act as
diluents, flavoring agents, binders, preservatives, and/or an
encapsulating material.
[0091] In another aspect of the present invention, the SDF-1 or
SDF-1 agent can be formulated for topical administration to treat
surface wounds. Topical formulations include those for delivery via
the mouth (buccal) and to the skin such that at least one layer of
skin (i.e., the epidermis, dermis, and/or subcutaneous layer) is
contacted with SDF-1 or agent. Topical delivery systems may be used
to administer topical formulations of the present invention.
[0092] Formulations for topical administration to the skin can
include ointments, creams, gels, and pastes comprising SDF-1 or
SDF-1 agent to be administered in a pharmaceutically acceptable
carrier. Topical formulations can be prepared using oleaginous or
water-soluble ointment bases, as is well known to those in the art.
For example, these formulations may include vegetable oils, animal
fats, and more preferably semisolid hydrocarbons obtained from
petroleum. Particular components used may include white ointment,
yellow ointment, cetyl esters wax, oleic acid, olive oil, paraffin,
petrolatum, white petrolatum, spermaceti, starch glycerite, white
wax, yellow wax, lanolin, anhydrous lanolin, and glyceryl
monostearate. Various water-soluble ointment bases may also be used
including, for example, glycol ethers and derivatives, polyethylene
glycols, polyoxyl 40 stearate, and polysorbates.
[0093] In another aspect of the invention, SDF-1 or agent can be
provided in and/or on a substrate, solid support, and/or wound
dressing for delivery of the SDF-1 or agent to the wound. As used
herein, the term "substrate," or "solid support" and "wound
dressing" refer broadly to any substrate when prepared for, and
applied to, a wound for protection, absorbance, drainage, etc. The
present invention may include any one of the numerous types of
substrates and/or backings that are commercially available,
including films (e.g., polyurethane films), hydrocolloids
(hydrophilic colloidal particles bound to polyurethane foam),
hydrogels (cross-linked polymers containing about at least 60%
water), foams (hydrophilic or hydrophobic), calcium alginates
(non-woven composites of fibers from calcium alginate), and
cellophane (cellulose with a plasticizer). The shape and size of a
wound may be determined and the wound dressing customized for the
exact site based on the measurements provided for the wound. As
wound sites can vary in terms of mechanical strength, thickness,
sensitivity, etc., the substrate can be molded to specifically
address the mechanical and/or other needs of the site. For example,
the thickness of the substrate may be minimized for locations that
are highly innervated, e.g., the fingertips. Other wound sites,
e.g., fingers, ankles, knees, elbows and the like, may be exposed
to higher mechanical stress and require multiple layers of the
substrate.
[0094] In one example, the substrate can be a bioresorbable implant
that includes a polymeric matrix and the SDF-1 or agent dispersed
in the matrix. The polymeric matrix may be in the form of a
membrane, sponge, gel, or any other desirable configuration. The
polymeric matrix can be formed from biodegradable polymer. It will
be appreciated, however, that the polymeric matrix may additionally
comprise an inorganic or organic composite. The polymeric matrix
can comprise any one or combination of known materials including,
for example, chitosan, poly(ethylene oxide), poly (lactic acid),
poly(acrylic acid), poly(vinyl alcohol), poly(urethane),
poly(N-isopropyl acrylamide), poly(vinyl pyrrolidone) (PVP), poly
(methacrylic acid), poly(p-styrene carboxylic acid),
poly(p-styrenesulfonic acid), poly(vinylsulfonicacid),
poly(ethyleneimine), poly(vinylamine), poly(anhydride),
poly(L-lysine), poly(L-glutamic acid), poly(gamma-glutamic acid),
poly(caprolactone), polylactide, poly(ethylene), poly(propylene),
poly(glycolide), poly(lactide-co-glycolide), poly(amide),
poly(hydroxylacid), poly(sulfone), poly(amine), poly(saccharide),
poly(HEMA), poly(anhydride), collagen, gelatin, glycosaminoglycans
(GAG), poly (hyaluronic acid), poly(sodium alginate), alginate,
hyaluronan, agarose, polyhydroxybutyrate (PHB), and the like.
[0095] It will be appreciated that one having ordinary skill in the
art may create a polymeric matrix of any desirable configuration,
structure, or density. By varying polymer concentration, solvent
concentration, heating temperature, reaction time, and other
parameters, for example, one having ordinary skill in the art can
create a polymeric matrix with any desired physical
characteristic(s). For example, the polymeric matrix may be formed
into a sponge-like structure of various densities. The polymeric
matrix may also be formed into a membrane or sheet which could then
be wrapped around or otherwise shaped to a wound. The polymeric
matrix may also be configured as a gel, mesh, plate, screw, plug,
or rod. Any conceivable shape or form of the polymeric matrix is
within the scope of the present invention. In an example of the
present invention, the polymeric matrix can comprise a alginate
matrix.
[0096] In another aspect of the present invention, at least one
progenitor cell can be provided in the polymeric matrix. Examples
progenitor cells can be selected from, but not restricted to,
totipotent stem cell, pluripotent stem cell, multipotent stem cell,
mesenchymal stem cell, neuronal stem cell, hematopoietic stem cell,
pancreatic stem cell, cardiac stem cell, embryonic stem cell,
embryonic germ cell, neural crest stem cell, kidney stem cell,
hepatic stem cell, lung stem cell, hemangioblast cell, and
endothelial progenitor cell. Additional examples of progenitor
cells can be selected from, but not restricted to,
de-differentiated chondrogenic cells, myogenic cells, osteogenic
cells, tendogenic cells, ligamentogenic cells, adipogenic cells,
and dermatogenic cells.
[0097] The polymeric matrix of the present invention may be seeded
with at least one progenitor cell and the SDF-1 or agent. The SDF-1
or agent can be dispersed in matrix and/or expressed from the
seeded progenitor cell. Progenitor cells can include autologous
cells; however, it will be appreciated that xenogeneic, allogeneic,
or syngeneic cells may also be used. Where the cells are not
autologous, it may be desirable to administer immunosuppressive
agents in order to minimize immunorejection. The progenitor cells
employed may be primary cells, explants, or cell lines, and may be
dividing or non-dividing cells. Progenitor cells may be expanded ex
vivo prior to introduction into the polymeric matrix. Autologous
cells are preferably expanded in this way if a sufficient number of
viable cells cannot be harvested from the host.
[0098] The SDF-1 or SDF-1 agent can also be provided in or on a
surface of a medical device used to treat an internal and/or
external wound. The medical device can comprise any instrument,
implement, machine, contrivance, implant, or other similar or
related article, including a component or part, or accessory, which
is, for example, recognized in the official U.S. National
Formulary, the U.S. Pharmacopoeia, or any supplement thereof; is
intended for use in the diagnosis of disease or other conditions,
or in the cure, mitigation, treatment, or prevention of disease, in
humans or in other animals; or, is intended to affect the structure
or any function of the body of humans or other animals, and which
does not achieve any of its primary intended purposes through
chemical action within or on the body of man or other animals, and
which is not dependent upon being metabolized for the achievement
of any of its primary intended purposes.
[0099] The medical device can include, for example, endovascular
medical devices, such as intracoronary medical devices. Examples of
intracoronary medical devices can include stents, drug delivery
catheters, grafts, and drug delivery balloons utilized in the
vasculature of a subject. Where the medical device comprises a
stent, the stent may include peripheral stents, peripheral coronary
stents, degradable coronary stents, non-degradable coronary stents,
self-expanding stents, balloon-expanded stents, and esophageal
stents. The medical device may also include arterio-venous grafts,
by-pass grafts, penile implants, vascular implants and grafts,
intravenous catheters, small diameter grafts, artificial lung
catheters, electrophysiology catheters, bone pins, suture anchors,
blood pressure and stent graft catheters, breast implants, benign
prostatic hyperplasia and prostate cancer implants, bone
repair/augmentation devices, breast implants, orthopedic joint
implants, dental implants, implanted drug infusion tubes,
oncological implants, pain management implants, neurological
catheters, central venous access catheters, catheter cuff, vascular
access catheters, urological catheters/implants, atherectomy
catheters, clot extraction catheters, PTA catheters, PTCA
catheters, stylets (vascular and non-vascular), drug infusion
catheters, angiographic catheters, hemodialysis catheters,
neurovascular balloon catheters, thoracic cavity suction drainage
catheters, electrophysiology catheters, stroke therapy catheters,
abscess drainage catheters, biliary drainage products, dialysis
catheters, central venous access catheters, and parental feeding
catheters.
[0100] The medical device may additionally include either
implantable pacemakers or defibrillators, vascular grafts,
sphincter devices, urethral devices, bladder devices, renal
devices, gastroenteral and anastomotic devices, vertebral disks,
hemostatic barriers, clamps, surgical
staples/sutures/screws/plates/wires/clips, glucose sensors, blood
oxygenator tubing, blood oxygenator membranes, blood bags, birth
control/IUDs and associated pregnancy control devices, cartilage
repair devices, orthopedic fracture repairs, tissue scaffolds, CSF
shunts, dental fracture repair devices, intravitreal drug delivery
devices, nerve regeneration conduits, electrostimulation leads,
spinal/orthopedic repair devices, wound dressings, embolic
protection filters, abdominal aortic aneurysm grafts and devices,
neuroaneurysm treatment coils, hemodialysis devices, uterine
bleeding patches, anastomotic closures, aneurysm exclusion devices,
neuropatches, vena cava filters, urinary dilators, endoscopic
surgical and wound drainings, surgical tissue extractors,
transition sheaths and dilators, coronary and peripheral
guidewires, circulatory support systems, tympanostomy vent tubes,
cerebro-spinal fluid shunts, defibrillator leads, percutaneous
closure devices, drainage tubes, bronchial tubes, vascular coils,
vascular protection devices, vascular intervention devices
including vascular filters and distal support devices and emboli
filter/entrapment aids, AV access grafts, surgical tampons, cardiac
valves, and tissue engineered constructs, such as bone grafts and
skin grafts.
[0101] The following examples are for the purpose of illustration
only and are not intended to limit the scope of the claims, which
are appended hereto.
Example 1
Stromal Cell-Derived Factor-1 Release in Alginate Scaffolds:
Characterization and Ability to Accelerate Wound Healing
[0102] We hypothesized that a slow-release delivery of either SDF-1
protein or plasmid would increase its effectiveness on wound
healing. Therefore, we employed a clinically-relevant delivery
system, an alginate scaffold, to deliver SDF-1 over time to a
porcine acute surgical wound model. We characterize SDF-1 delivery
using alginate scaffolds in vitro, and demonstrated the potential
for therapeutic benefit in vivo by using the scaffolds to deliver
SDF-1 protein and plasmid to acute surgical wounds.
Preparation of Scaffolds for In Vivo Application
[0103] For the in vivo application, custom 1 cm.times.6 cm alginate
scaffolds were produced by the same process described above.
Scaffolds were then loaded with SDF-1 plasmid (n=6), SDF-1 protein
(n=10), or phosphate buffered saline (PBS) (n=4) by the process
described below.
[0104] For the SDF-1 plasmid scaffolds, a plasmid was created by
inserting the gene encoding human SDF-1 in a pcDNA3.1 backbone
(Invitrogen Corporation, Carlsbad, Calif.). A loading solution was
prepared by mixing 3.5 mg of the SDF-1 plasmid in 2.33 ml PBS to
create a 1.5 mg/ml solution. On each scaffold, the loading solution
was pipetted under sterile conditions onto the scaffold in six 60
.mu.l drops (360 .mu.l total) equally spaced so that each drop
covered a 1 cm.times.1 cm area of the scaffold.
[0105] For the SDF-1 protein scaffolds, a loading solution was
prepared by mixing 10 .mu.g of carrier-free SDF-1 protein (R&D
systems, Minneapolis, Minn.) with 5 mL PBS and 3 ml of 1000 IU/ml
injection heparin (Baxter Healthcare Corporation, Deerfield, Ill.)
to create a 1.5 .mu.g/ml solution. On each scaffold, the loading
solution was pipetted under sterile conditions onto the scaffold in
six equally spaced 60 .mu.l drops.
[0106] The PBS scaffolds served as a negative control. The loading
solution was prepared by mixing 1.35 mL PBS and 0.45 ml of 1000
IU/ml injection heparin. The loading solution was pipetted under
sterile conditions onto the scaffold in six equally spaced 60 .mu.l
drops.
[0107] All loaded scaffolds were stored at 4.degree. C. for 12
hours prior to applying them to the wounds.
Porcine Surgical Wound Healing Model and Ante-Mortem Follow-Up
[0108] In 2 Domestic Yorkshire pigs, general anesthesia was
induced. A cuffed endotracheal tube was placed and general
anesthesia was maintained with isoflurane delivered in oxygen
through a rebreathing system with ventilator assist. A standard
model of acute surgical wounds was used. Each animal received
twelve (12) 5 cm full thickness incisions (six on each side of the
spine) spaced approximately 7.5 cm apart. Each incision was made
perpendicular to the spine, starting 7.5 cm from the spine and
cutting toward the abdomen. Gauze was placed in the incision until
the bleeding stopped. The gauze was removed, and the incision was
sutured closed.
[0109] Following wound closure, the scaffold was placed next to the
wound and photographed (FIG. 1). On each pig, the scaffold
placement order was randomized with the following distribution:
[0110] SDF-1 protein scaffold (n=5) [0111] SDF-1 plasmid scaffold
(n=3) [0112] PBS scaffold (control, n=2) [0113] No scaffold (sham,
n=2)
[0114] The scaffold was placed over the wound (except in the sham
group), and each wound was dressed with a Tegaderm.TM. patch.
[0115] To determine the effect of SDF-1 on the rate of wound
healing, wound length was measured by the same veterinarian at day
0 (prior to scaffold placement) and prior to sacrifice. Wound
length was converted to Percent Healing by the following
relationship:
(Initial wound length-final wound length)/initial wound
length*100%
[0116] To monitor both the acute and chronic effects of SDF-1 on
wound healing, the acute effects were evaluated in the first pig,
which was sacrificed at 4 days, and the chronic effects in the
second which was sacrificed at 9 days.
Post-Mortem Follow-Up
[0117] Following sacrifice, one section from the middle of each
wound site was excised for histopathological and
immunohistochemical analysis. Standard hematoxylin and eosin
(H&E) stain was used to assess extent of fibroplasia,
inflammation, and necrosis at day 4 and necrosis, fibrosis, and
granulomatous inflammation at day 9. Each parameter was graded on a
qualitative scale by a histopathologist blinded to randomization as
either: none (not present), minimal, mild, moderate, or severe.
Immunohistochemical staining was performed on the same tissue
section. The effect of SDF-1 on fibroblast infiltration into the
wound was detected by vimentin staining. The effect on blood vessel
formation was determined by CD31 and the presence of smooth muscle
was detected by smooth muscle actin staining. The amount of each
stain per sample was graded by the same pathologist using the same
qualitative scale as above (minimal severe).
[0118] The impact of an SDF-1-releasing scaffold on wound healing
is also shown in FIGS. 1 and 2. FIG. 1 shows representative
examples of wounds treated with control (PBS) scaffold, SDF-1
protein scaffold, and SDF-1 plasmid scaffold at day 0 (top panel)
and day 9 (bottom panel). All full-incision wounds (middle) have a
length 5.0.+-.0.1 cm.
[0119] At day 9, the wound treated with the control scaffold is
still apparent, and has a Percent Healed of 0%. In contrast, both
the SDF-1 protein and SDF-1 plasmid treated wounds are no longer
visible at day 9, and both have a Percent Healed of 100%.
[0120] FIG. 2 summarizes the percent healing data for all treated
wounds. Day 4 data is from the first pig, and Day 9 data is from
the second pig. At Day 9, the wounds treated with either the SDF-1
plasmid or protein scaffolds (solid markers and lines) have healed
to a greater extent than the control or sham groups (open markers
and dotted lines). Notably, 1 of 3 SDF-1 plasmid treated wounds and
2 of 5 SDF-1 protein treated wounds are 100% healed at 9 days;
whereas, no control or sham wound are greater than 20% healed at 9
days.
[0121] We investigated the impact of SDF-1 on fibroblast
infiltration, new blood vessel formation, and smooth muscle using
immunohistochemical staining for vimentin, CD31, and smooth muscle
actin, respectively. There are no substantial differences in amount
of any of the stains between groups. H & E analysis showed a
slight decrease in fibrosis in the SDF-1 protein and plasmid
treated wounds compared to control or sham, with all other
parameters being similar. The results are shown below in the
following tables.
[0122] The results are shown the table below.
TABLE-US-00001 Wound Healing H/E data - Day 9 # wounds with
fibrosis Sham (no patch) 2 1 1 0 2 (of 2) 50% Control (saline
patch) 2 1 0 1 2 (of 2) 50% SDF1 Protein Patch 5 4 1 0 5 (of 5) 80%
SDF1 Plasmid Patch 3 3 0 0 3 (of 3) 100% # wounds with
granulomatous inflammation Sham (no patch) 2 0 0 0 (of 2) 50%
Control (saline patch) 2 0 1 1 (of 2) 50% SDF1 Protein Patch 5 1 0
1 (of 5) 80% SDF1 Plasmid Patch 3 0 0 0 (of 3) 100% # of wounds
with necrosis Sham (no patch) 2 1 0 1 (of 2) Control (saline patch)
2 0 0 0 (of 2) SDF1 Protein Patch 5 1 1 2 (of 5) SDF1 Plasmid Patch
3 1 0 1 (of 3) # of wounds with sub-acute inflammation Sham (no
patch) 2 1 1 0 2 (of 2) Control (saline patch) 2 0 0 0 0 (of 2)
SDF1 Protein Patch 5 0 1 1 2 (of 5) SDF1 Plasmid Patch 3 1 0 1 2
(of 3)
[0123] From the above description of the invention, those skilled
in the art will perceive improvements, changes and modifications.
Such improvements, changes and modifications within the skill of
the art are intended to be covered by the appended claims. All
patents, patent applications and publications cited herein are
incorporated by reference in their entirety.
Sequence CWU 1
1
5168PRTHomo sapiens 1Lys Pro Val Ser Leu Leu Tyr Arg Cys Pro Cys
Arg Phe Phe Glu Ser 1 5 10 15 His Val Ala Arg Ala Asn Val Lys His
Leu Lys Ile Leu Asn Thr Pro 20 25 30 Asn Cys Ala Leu Gln Ile Val
Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45 Val Cys Ile Asp Pro
Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys 50 55 60 Ala Leu Asn
Lys 65 289PRTHomo sapiens 2Met Asn Ala Lys Val Val Val Val Leu Val
Leu Val Leu Thr Ala Leu 1 5 10 15 Cys Leu Ser Asp Gly Lys Pro Val
Ser Leu Ser Tyr Arg Cys Pro Cys 20 25 30 Arg Phe Phe Glu Ser His
Val Ala Arg Ala Asn Val Lys His Leu Lys 35 40 45 Ile Leu Asn Thr
Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys 50 55 60 Asn Asn
Asn Arg Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln 65 70 75 80
Glu Tyr Leu Glu Lys Ala Leu Asn Lys 85 389PRTRattus norvegicus 3Met
Asp Ala Lys Val Val Ala Val Leu Ala Leu Val Leu Ala Ala Leu 1 5 10
15 Cys Ile Ser Asp Gly Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys
20 25 30 Arg Phe Phe Glu Ser His Val Ala Arg Ala Asn Val Lys His
Leu Lys 35 40 45 Ile Leu Asn Thr Pro Asn Cys Ala Leu Gln Ile Val
Ala Arg Leu Lys 50 55 60 Ser Asn Asn Arg Gln Val Cys Ile Asp Pro
Lys Leu Lys Trp Ile Gln 65 70 75 80 Glu Tyr Leu Asp Lys Ala Leu Asn
Lys 85 41940DNAHomo sapiens 4gccgcacttt cactctccgt cagccgcatt
gcccgctcgg cgtccggccc ccgacccgcg 60ctcgtccgcc cgcccgcccg cccgcccgcg
ccatgaacgc caaggtcgtg gtcgtgctgg 120tcctcgtgct gaccgcgctc
tgcctcagcg acgggaagcc cgtcagcctg agctacagat 180gcccatgccg
attcttcgaa agccatgttg ccagagccaa cgtcaagcat ctcaaaattc
240tcaacactcc aaactgtgcc cttcagattg tagcccggct gaagaacaac
aacagacaag 300tgtgcattga cccgaagcta aagtggattc aggagtacct
ggagaaagct ttaaacaagt 360aagcacaaca gccaaaaagg actttccgct
agacccactc gaggaaaact aaaaccttgt 420gagagatgaa agggcaaaga
cgtgggggag ggggccttaa ccatgaggac caggtgtgtg 480tgtggggtgg
gcacattgat ctgggatcgg gcctgaggtt tgccagcatt tagaccctgc
540atttatagca tacggtatga tattgcagct tatattcatc catgccctgt
acctgtgcac 600gttggaactt ttattactgg ggtttttcta agaaagaaat
tgtattatca acagcatttt 660caagcagtta gttccttcat gatcatcaca
atcatcatca ttctcattct cattttttaa 720atcaacgagt acttcaagat
ctgaatttgg cttgtttgga gcatctcctc tgctcccctg 780gggagtctgg
gcacagtcag gtggtggctt aacagggagc tggaaaaagt gtcctttctt
840cagacactga ggctcccgca gcagcgcccc tcccaagagg aaggcctctg
tggcactcag 900ataccgactg gggctgggcg ccgccactgc cttcacctcc
tctttcaacc tcagtgattg 960gctctgtggg ctccatgtag aagccactat
tactgggact gtgctcagag acccctctcc 1020cagctattcc tactctctcc
ccgactccga gagcatgctt aatcttgctt ctgcttctca 1080tttctgtagc
ctgatcagcg ccgcaccagc cgggaagagg gtgattgctg gggctcgtgc
1140cctgcatccc tctcctccca gggcctgccc cacagctcgg gccctctgtg
agatccgtct 1200ttggcctcct ccagaatgga gctggccctc tcctggggat
gtgtaatggt ccccctgctt 1260acccgcaaaa gacaagtctt tacagaatca
aatgcaattt taaatctgag agctcgcttt 1320gagtgactgg gttttgtgat
tgcctctgaa gcctatgtat gccatggagg cactaacaaa 1380ctctgaggtt
tccgaaatca gaagcgaaaa aatcagtgaa taaaccatca tcttgccact
1440accccctcct gaagccacag cagggtttca ggttccaatc agaactgttg
gcaaggtgac 1500atttccatgc ataaatgcga tccacagaag gtcctggtgg
tatttgtaac tttttgcaag 1560gcattttttt atatatattt ttgtgcacat
ttttttttac gtttctttag aaaacaaatg 1620tatttcaaaa tatatttata
gtcgaacaat tcatatattt gaagtggagc catatgaatg 1680tcagtagttt
atacttctct attatctcaa actactggca atttgtaaag aaatatatat
1740gatatataaa tgtgattgca gcttttcaat gttagccaca gtgtattttt
tcacttgtac 1800taaaattgta tcaaatgtga cattatatgc actagcaata
aaatgctaat tgtttcatgg 1860tataaacgtc ctactgtatg tgggaattta
tttacctgaa ataaaattca ttagttgtta 1920gtgatggagc ttaaaaaaaa
19405293DNARattus norvegicus 5ccatggacgc caaggtcgtc gctgtgctgg
ccctggtgct ggccgcgctc tgcatcagtg 60acggtaagcc agtcagcctg agctacagat
gcccctgccg attctttgag agccatgtcg 120ccagagccaa cgtcaaacat
ctgaaaatcc tcaacactcc aaactgtgcc cttcagattg 180ttgcaaggct
gaaaagcaac aacagacaag tgtgcattga cccgaaatta aagtggatcc
240aagagtacct ggacaaagcc ttaaacaagt aagcacaaca gcccaaagga ctt
293
* * * * *