Note: Descriptions are shown in the official language in which they were submitted.
CA 02758178 2011-11-07
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THIS IS VOLUME 1 OF 2
NOTE: For additional volumes please contact the Canadian Patent Office.
CA 02758178 2011-11-07
HUMAN SIUN EQUIVALENTS EXPRESSING EXOGENOUS POLYPEPTIDES
This application was supported in part by STTR. Fast-Track Grant Phase I #1
R41
AR 0530349-01 and Phase B. #14 R42 AR 050349-02. The government may have
certain
rights in the invention.
FIELD OF THE INVENTION
The present invention relates generally to compositions for wound closure.
More
specifically, the present invention provides human skin equivalents engineered
to express
exogenous polypeptides (e.g., antimicrobial polypeptides and keratinoeyte
growth factor 2)
.and compositions and methods for making human skin equivalents engineered to
express
exogenous polypeptides. In addition, the present invention provides methods
for treatment
of wounds with human skin equivalents engineered to express exogenous
polypeptides.
BACKGROUND
Clu-onic wounds affect three million people each year in the U.S. Chronic
wounds
generally involve any break, or ulceration, of the skin that is of long
duration or recurs
frequently. Such wounds cause pain, loss of function, force changes in an
individual's life
through potential lack of mobility, take extended periods of time for
recovery, and require
high amounts of patient conipliance for recovery.
Chronic wounds disrupt the integrity of the skin by tearing, cutting, piercing
or
breaking the tissue. The Causes may be structural, such as injury, or
physiological, such as
an underlying disease. The most frequently occurring skin wounds are venous
ulcers,
pressure ulcers and diabetic foot ulcers.
Chronic wounds are a serious health concern with substantial morbidity. They
also
are a source of frustration to both physician and patient, as lengthy
treatments, treatment
failures and the need for long periods of patient compliance prove
challenging. The wounds
take so long to heal that conipliance drops off and worsens when reversals
occur or new
ulcers appear.
Chronic wounds occiu in individuals with underlying diseases of various types
whose medical conditions compromise the body's ability to repair injured
tissue on its own.
Despite the use of a variety of medical and surgical treatments, chronic
wounds can take
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months or even years to heal and frequently recur. These wounds are often
large and
unsightly and may be painful in some patients.
Chronic wounds are of three major types: venous stasis ulcers, diabetic ulcers
and
pressure ulcers. A venous ulcer is an ulceration that develops on the ankle or
lower leg in
patients with chronic vascular disease. In these patients, blood flow in the
lower extremities
is impaired, leading to edema (swelling) and mild redness and scaling of the
skin that
gradually progress to ulceration. Venous ulcers are a condition affecting
500,000 - 700,000
patients in the US and 1.3 million people in the industrialized world.
A diabetic ulcer is a chronic wound that occurs in patients with diabetes.
While the
actual cause of the ulcer in these patients is an injury such as a callus,
blister or foreign body
such as a pebble or splinter, it is the patient's underlying disease that
places him or her at
high risk for developing an ulcer. Important risk factors include: inadequate
local blood
supply, which impairs their ability to repair injured tissue and ward off
infection, and
reduced sensation in the extremities, which causes the initial injury to go
unrecognized until
it becomes a serious, chronic wound. Diabetic ulcers are a condition affecting
just under
500,000 patients in the US and 1.2 million people in the industrialized world.
A pressure ulcer is defined as any lesion caused by unrelieved pressure on
tissues
that are located over a bony prominence on the body. Pressure ulcers were
formerly referred
to as bedsores or decubitus ulcers. Pressure ulcers develop in immobile
patients whose
tissues are subjected to continuous pressure from bones on the interior and
hard surfaces
such as beds or chairs on the exterior. In addition to their immobility,
patients at risk for the
development of pressure ulcers typically have poor nutritional status,
inadequate hydration,
and other underlying medical conditions that compromise their ability to heal
injuries.
Pressure ulcers affect over 1.6 million people in the US and 4.1 million
people in the
industrialized world. Estimates of the prevalence of these conditions vary
greatly.
Estimates as high as 12 million patients have been reported for all types of
chronic wounds
in the industrialized markets.
Chronic wounds can be of variable sizes and depths. In general, there are four
layers
of tissue that can potentially sustain injury in a wound, the epidermis, or
outermost layer;
the dermis; the subcutaneous tissue; and, at the deepest layer, muscle,
tendon, and bone.
Partial-thickness ulcers involve a loss of skin that is limited to the
epidermis and,
potentially, part of the dermis, These wounds heal by epithelialization
(proliferation and
migration of epithelial cells). Full-thickness ulcers involve damage or
necrosis of the
epidermis, dermis, and subcutaneous tissue, and may extend into the connective
tissue
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below the dermis. These wounds heal by granulation (filling of the wound with
connective
tissue), contraction, and epithelialization. The most severe category of ulcer
involves injury
to the epidermis, dermis, subcutaneous tissue, and muscle, tendon, or bone.
The wound
healing process is not complete even after the wound has closed. The process
of rebuilding
normal skin and tissue in a wound can take up to two years after the initial
injury.
Treatment of chronic wounds varies with the severity of the wound. Partial-
and full-
thickness wounds are typically treated with dressings and debridement (use of
chemicals or
surgery to clear away necrotic, or dead, tissue). Antibiotics may be used in
the event of an
infection. Partial-thickness to fall-thickness wounds represent the largest
categories of
chronic wound patients, the areas of greatest unmet medical need, and the
categories most
amenable to treatment with prescription growth factor therapy such as
Repifermin. Patients
with full-thickness wounds extending into muscle, tendon or bone are at
significant risk of
sepsis and are typically treated with surgery.
Despite the number of conservative therapies available, chronic wounds remain
a
very frustrating problem for health care practitioners because of the time-
consuming nature
of treatment regimens and patient non-compliance. What is needed is a therapy
that can
increase a practitioner's success in healing chronic wounds and/or accelerate
the rate of
chronic wound healing.
SUMMARY OF THE INVENTION
The present invention relates generally to compositions for wound closure.
More
specifically, the present invention provides human skin equivalents engineered
to express
exogenous polypeptides (e.g., antimicrobial polypeptides and keratinocyte
growth factor 2)
and compositions and methods for making human skin equivalents engineered to
express
exogenous polypeptides. In addition, the present invention provides methods
for treatment
of wounds with human skin equivalents engineered to express exogenous
polypeptides.
Accordingly, in some embodiments, the present invention provides methods for
providing cells expressing heterologous KGF-2 comprising: a) providing a host
cell selected
from the group consisting of primary keratinocytes and immortalized
keratinocytes and an
expression vector comprising a DNA sequence encoding KGF-2 operably linked to
a
regulatory sequence; b) introducing the expression vector to the host cell
(e.g., under
conditions such that said expression vector is internalized by the host cell);
and c) culturing
the host cells under conditions such that KGF-2 is expressed. The present
invention is not
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limited to the use of any particular primary or immortalized keratinocytes. In
some
preferred embodiments, the keratinocytes are NIKS cells or cell derived from
NIKS cells.
In other embodiments, the keratinocytes are capable of stratifying into
squamous epithelia.
In still other embodiments, the methods include the step of co-culturing the
host cells with
cells derived from a patient. The present invention is not limited to the use
of any particular
expression vector. In some embodiments, the expression vector further
comprises a
selectable marker. The present invention is not limited to the use of any
particular
regulatory sequence. In some embodiments, the regulatory sequence is a
promoter
sequence. The present invention is not limited to any particular promoter
sequence. In
some embodiments, the promoter sequence is K14 promoter sequence, preferably a
full-
length K14 promoter sequence. In other embodiments, the promoter is an
involucrin
promoter. In preferred embodiments, the promoter sequence allows expression in
a
keratinocyte. In still further embodiments, the present invention provides
host cells
produced by the foregoing method.
In some embodiments, the present invention provides compositions comprising
host
cells expressing heterologous KGF-2, wherein the host cells are selected from
the group
consisting of primary and immortalized keratinocytes. In some embodiments, the
host cells
are NIKS cells or cell derived from NIKS cells. In further embodiments, the
KGF-2 is full
length KGF-2.
In further embodiments, the present invention provides methods of treating
wounds
comprising: a) providing immortalized keratinocytes expressing heterologous
KGF-2, and a
subject with a wound; and b) contacting the wound with the immortalized cells
expressing
heterologous KGF-2. The present invention is not limited to any particular
type of
contacting. Indeed, a variety of ways of contacting are contemplated. In some
embodiments, the contacting comprises topical application. In other
embodiments, the
contacting comprises engraftment. In still other embodiments, the contacting
comprises
wound dressing. The present invention is not limited to the treatment of any
particular type
of wound. Indeed, the treatment of a variety of wounds is contemplated,
including, but not
limited to those selected from the group comprising venous ulcers, diabetic
ulcers, pressure
ulcers, burns, ulcerative colitis, mucosal injuries, internal injuries,
external injuries. In
some embodiments, the immortalized keratinocytes are MKS cells. In further
embodiments, the Munortalized keratinocytes are incorporated into a human skin
equivalent. In still further embodiments, the human skin equivalent further
comprises cells
derived from a patient. In other embodiments, the methods further comprise the
step
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mixing the keratinocytes expressing heterologous KGF-2 with cells derived from
the subject
prior to the contacting step.
In still other embodiments, the present invention provides vectors comprising
a
keratinocyte specific promoter operably linked to a DNA sequence encoding KGF-
2. In
some embodiments, the keratinocyte specific promoter is the K14 promoter or
the
involucrin promoter. The present invention also provides host cells and skin
equivalents
comprising these vectors.
In other embodiments, the present invention provides a method for providing a
tissue (e.g., human skin equivalent) expressing an exogenous antimicrobial
polypeptide or
peptide comprising providing a keratinocyte and an expression vector
comprising a DNA
sequence encoding an antimicrobial polypeptide or peptide thereof operably
linked to a
regulatory sequence; introducing the expression vector into the keratinocyte;
and
incorporating the keratinocyte into a tissue (e.g., human skin equivalent). In
some
embodiments, the keratinocyte is capable of stratifying into squamous
epithelia. In some
embodiments, the keratinocyte is selected a primary or immortalized
keratinocyte (e.g.
preferably NIKS cells). In certain embodiments, the expression vector further
comprises a
selectable marker. In some preferred embodiments, the regulatory sequence is a
promoter
sequence (e.g., an involucrin promoter or a keratin-I4 promoter). In certain
preferred
embodiments, the promoter sequence allows antimicrobial polypeptide expression
in the
host cell. The present invention is not limited to a particular antimicrobial
polypeptide.
Indeed, a variety of antimicrobial polypeptides is contemplated including, but
not limited to,
human beta defensin 1, 2, and 3 and human cathelicidin. In some embodiments,
the human
beta defensin 3 has a mutated amino acid sequence (e.g., one or more single
amino acid
substitutions). In some preferred embodiments, the one or more single amino
acid
substitutions comprise Cys40Ala, Cys45Ala, Cys55Ala, Cys62Ala, and Cys63Ala.
In other
em.bodiments, the single amino acid substitution is Gly38Ala. In particularly
preferred
embodiments, the mutated human beta defenin 3 has antimicrobial activity. In
other
embodiments, the expression vector further comprises a nucleic acid sequence
encoding a
signal secretion peptide. In preferred embodiments, the skin equivalent
exhibits
antimicrobial activity. The present invention additionally provides a skin
equivalent
produced by the method described herein.
In yet other embodiments, the present invention provides a composition
comprising
keratinocytes (e.g., primary or immortalized keratinocytes) expressing an
exogenous
antimicrobial polypeptide. In preferred embodiments, the keratinocytes are
NTKS cells or
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cells derived from NIKS cells. The present invention is not limited to a
particular
antimicrobial polypeptide. Indeed, a variety of antimicrobial polypeptides is
contemplated
including, but not limited to, human beta defensin 1, 2, and 3 and human
cathelicidin. In
some embodiments, the human beta defensin 3 has a mutated amino acid sequence
(e.g.,
one or more single amino acid substitutions). In some preferred embodiments,
the one or
more single amino acid substitutions comprise Cys40Ala, Cys45Ala, Cys55Ala,
Cys62Ala,
and Cys63Ala. In other embodiments, the single amino acid substitution is
Gly38Ala. In
some embodiments, the keratinocytes are stratified. In other embodiments, the
composition
further comprises a demial equivalent. In yet other embodiments, the present
invention
provides an organotypie culture of the keratinocytes. In other embodiments,
the
composition further comprises cells derived from a patient. In still further
embodiments,
the composition further comprises keratinocytes that do not express the
exogenous
antimicrobial polyeptide. In yet other embodiments, the composition further
comprises
keratinocytes expressing at least one additional exogenous (e.g.,
antimicrobial) polypeptide.
The present invention further provides a method of treating wounds comprising:
providing primary or immortalized keratinocytes (e.g., NIKS cells) expressing
a exogenous
antimicrobial polypeptide, and a subject with a wound; contacting the wound
with the
immortalized keratinocytes expressing an exogenous antimcrobial polypeptide.
The present
invention is not limited to a particular antimicrobial polypeptide. Indeed, a
variety of
antimicrobial polypeptides is contemplated including, but not limited to,
human beta
defensin 1, 2, and 3 and human cathelicidin. In some embodiments, the human
beta
defensin 3 has a mutated amino acid sequence (e.g., one or more single amino
acid
substitutions). In some preferred embodiments, the one or more single amino
acid
substitutions comprise Cys40Ala, Cys45Ala, Cys55Ala, Cys62Ala, and Cys63Ala.
In other
embodiments, the single amino acid substitution is Gly38Ala. In some
embodiments, the
contacting comprises engraftment, topical application, or wound dressing. The
present
invention contemplates treatment of any type of wound, including, but not
limited to,
venous ulcers, diabetic ulcers, pressure ulcers, burns, ulcerative colitis,
mucousal injuries,
internal injuries, and external injuries. In some embodiments, the human skin
equivalent
further comprises cells derived from a patient.
The present invention additionally provides a vector comprising a keratinocyte
specific promoter (e.g., involucrin promoter or the keratin-14 promoter)
operably linked to a
DNA sequence encoding an antimicrobial polypeptide. The present invention is
not limited
to a particular antimicrobial polypeptide. Indeed, a variety of antimicrobial
polypeptides is
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contemplated including, but not limited to, human beta defensin 1, 2, and 3
and human
cathelicidin. In some embodiments, the human beta defensin 3 has a mutated
amino acid
sequence (e.g., one or more single amino acid substitutions). The present
invention further
provides a host cell comprising the vector. The present invention also
provides a human
tissue (e.g., skin equivalent) comprising the host cell. In some embodiments,
the human
tissue (e.g., skin equivalent) further comprises cells derived from a patient.
In other
embodiments, the human tissue (e.g., skin equivalent) further comprises
keratinocytes not
comprising the vector. In yet other embodiments, the human skin equivalent
further
comprises keratinocytes expressing at least one additional antimicrobial
polypeptide.
In yet other embodiments, the present invention provides a method for
providing a
human tissue (e.g., skin equivalent) expressing an exogenous KGF-2 and an
exogenous
antimicrobial polypeptide comprising providing a keratinocyte; a first
expression vector
comprising a DNA sequence encoding an antimicrobial polypeptide operably
linked to a
regulatory sequence; and a second expression vector comprising a DNA encoding
an
exogenous KGF-2 polypeptide; and introducing the expression vector into the
keratinocyte;
and incorporating the keratinocyte into a human tissue (e.g., skin
equivalent).
In still other embodiments, the present invention provides a method of
selecting cells
with increased pluripotency or multipotency relative to a population,
comprising providing
a population of cells; electroporating the cells under conditions such that
electroporated
cells with increased pluripotency or multipotency relative to the population
of cells are
selected. In some embodiments, the electroporated cells exhibit stern cell
like properties. In
some embodiments, the population of cells are keratinocytes and the
electroporated
keratinocytes have holoclone or meroclone cell morphology. In other
embodiments, the
electroporated cells exhibit extended proliferative capacity. In some
embodiments, the
population of cells is electroporated with an exogenous nucleic acid
expressing a selectable
marker. In certain embodiments, the method further comprises the step of
culturing the
cells under conditions such that only cells expressing the selectable marker
are selected for.
The present invention additionally provides a cell or population of cells
generated by the
method.
In certain embodiments, the present invention provides a method of selecting
keratinocytes with holoclone or meroclone cell morphology, comprising
providing a
population of keratinocytes; and electroporating the keratinocytes under
conditions such
that electroporated keratinocytes with holoclone or meroclone cell morphology
are selected.
In some embodiments, the holoclone cell morphology comprises one or more
properties
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selected from the group consisting of tightly packed cells, cells uniform in
size, colonies
with smooth colony edges, and an overall round colony morphology. In some
embodiments, the population of keratinocytes is electroporated with an
exogenous nucleic
acid expressing a selectable marker. In certain embodiments, the method
further comprises
the step of culturing the keratinocytes under conditions such that only cells
expressing the
selectable marker are selected for. The present invention also provides a
keratinocyte
population generated by the method.
A method for providing tissues expressing heterologous KGF-2 and/or
antimicrobial
polypeptide comprising providing a tissue and an expression vector comprising
a DNA
sequence encoding KGF-2 and/or antimicrobial polypeptide operably linked to a
regulatory
sequence; introducing said expression vector to said tissue under conditions
such that said
expression vector is internalized by a host cell contained in said tissue and
said KGF-2
and/or antimicrobial polypeptide is expressed. In some embodiments, the tissue
is a human
tissue (e.g., a human skin equivalent). In some embodiments, the expression
vector is
introduced to the tissue by particle bombardment, electroporation, or
transfection.
DESCRIPTION OF THE FIGURES
Figure 1 provides the consensus sequence of the K14 promoter.
Figure 2 provides a diagram of the construction of the K14-luciferase vector.
Figure 3 provides a diagram of the K14-KGF-2 vector.
Figure 4 provides a diagram of the RT-PCR strategy.
Figure 5 provides a diagram of a vector for the expression of KGF-2 by the
Involucrin promoter.
Figure 6 provides the DNA sequence for human beta defensin 1 (SEQ ID NO:9).
Figure 7 provides the DNA sequence for human beta defensin 2 (SEQ ID NO:10).
Figure 8 provides the DNA sequence for human beta defensin 3 (SEQ ID NO:11).
Figure 9 provides the DNA sequence for the involucrin promoter (SEQ ID NO:
12).
Figure 10 provides amino acid sequence alignments of the human P-defensins 1-3
Figure 11 is a schematic drawing demonstrating characteristic I3-defensin
covalent
cysteine disulfide bond formation.
Figure 12 is a restriction enzyme map of the human I3-defensin-1 mammalian
expression
vector.
Figure 13 provides the cloning strategy for the human P-defensin vectors.
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Figure 14 describes expression vectors for expression of human I3-defensin.
Figure 15 provides the results of a RT-PCR assay for expression of human 13-
defensin
mRNA.
Figure 16 provides the results of immunoblot detection of human P-defensin
protein.
Figure 17 shows the antimicrobial activity of human p-defensins 1, 2, and 3.
Figure 18 shows the antimicrobial activity of human ii-defensins 3 in an
organotypic
culture.
Figure 19 shows a linear map and restriction digest analysis of the hCAP18
vector.
Figure 20 shows the results of a RT-PCR assay for expression of hCAP18.
DEFINTIONS
As used herein, the term "growth factor" refers to extracellular molecules
that bind
to a cell-surface triggering an intracellular signaling pathway leading to
proliferation,
differentiation, or other cellular response. Examples of growth factors
include, but are not
limited to, growth factor I, trophic factor, Ca2+, insulin, hormones,
synthetic molecules,
pharmaceutical agents, and LDL.
As used herein, the term "keratinocyte growth factor" or "KGF" refers to a
member
of a group of structurally-distinct proteins known as FGFs that display
varying degrees of
sequence homology, suggesting that they are encoded by a related family of
genes. The
FGFs share common receptor sites on cell surfaces. KGF, for example, can bind
to FGFR-
3.
As used herein, the term "antimicrobial polypeptide" refers to polypeptides
and
peptides thereof that inhibit the growth of microbes (e.g., bacteria).
Examples of
antimicrobial polypeptides include, but are not limited to, the polypeptides
described in
Table 1 below (e.g., defensins or cathelicidins). Antimicrobial polypeptides
include
peptides synthesized from both L-amino and D-amino acids. "Antimicrobial
polypeptides"
also include peptide portions of the antimicrobial polypeptide, obtained by
any method
(e.g., synthesized or enzymatically obtained).
As used herein, the term "defensin" refers to a family of highly cross-linked,
structurally homologous antimicrobial peptides that are generally, but not
necessarily, found
in the azurophil granules of polymorphonuclear leukocytes (PMN's) with
homologous
peptides being present in macrophages.
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As used herein, the terms "human beta-defensin I" or "hBD1", when used in
reference to a protein or nucleic acid refers to a protein or nucleic acid
encoding a protein
that shares greater than about 50% identity with SEQ ID NO: 13 and also has at
least one
activity of wild type hBD1. Thus, the term hBD1 protein encompasses both
proteins that
are identical to wild-type hBD1 protein and those that are derived from wild
type hBD1
protein (e.g., variants of hBD1 protein or chimeric genes constructed with
portions of hBD1
protein coding regions).
As used herein, the term "activity of hBD1" refers to any activity of wild
type hBD1
protein (e.g., antimicrobial activity). The term is intended to encompass all
activities of
liliD1 protein, alone or in combination.
In particular, the term "IBD1 gene" refers to the full-length hBD1 nucleotide
sequence (e.g., contained in SEQ ID NO:9). However, it is also intended that
the term
encompass fragments of the hBD1 sequence, as well as other domains within the
full-length
hBD1 nucleotide sequence, as well as variants of hBD1. Furthermore, the terms
" hBD1
gene nucleotide sequence" or " hBD1 gene polynucleotide sequence" encompasses
DNA,
cDNA, and RNA (e.g., mRNA) sequences.
As used herein, the terms "human beta-defensin 2" or "hBD2", when used in
reference to a protein or nucleic acid refers to a protein or nucleic acid
encoding a protein
that shares greater than about 50% identity with SEQ ID NO:14 and also has at
least one
activity of wild type hBD2. Thus, the term hBD2 protein encompasses both
proteins that
are identical to wild-type hBD2 protein and those that are derived from wild
type hBD2
protein (e.g., variants of hBD2 protein or chimeric genes constructed with
portions of hBD2
protein coding regions).
As used herein, the tem "activity of hBD2" refers to any activity of wild type
hBD2
protein (e.g., antimicrobial activity). The term is intended to encompass all
activities of
hBD2 protein, alone or in combination.
In particular, the term "hBD2 gene" refers to the full-length hI3D1 nucleotide
sequence (e.g., contained in SEQ ID NO:10). However, it is also intended that
the term
encompass fragments of the hBD1 sequence, as well as other domains within the
full-length
hBD2 nucleotide sequence, as well as variants of hBD1. Furthermore, the terms
" hBD2
gene nucleotide sequence" or " hBD1 gene polymicleotide sequence" encompasses
DNA,
cDNA, and RNA (e.g., mRNA) sequences.
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As used herein, the terms "human beta-defensin 3" or "hBD3", when used in
reference to a protein or nucleic acid refers to a protein or nucleic acid
encoding a protein
that shares greater than about 50% identity with SEQ ID NO:15 and also has at
least one
activity of wild type hBD3. Thus, the term hBD3 protein encompasses both
proteins that
are identical to wild-type hBD3 protein and those that are derived from wild
type hBD3
protein (e.g., variants of hBD3 protein or chimeric genes constructed with
portions of hBD3
protein coding regions).
As used herein, the term "activity of hBD3" refers to any activity of wild
type hBD3
protein (e.g., antimicrobial activity). The tern is intended to encompass all
activities of
hBD1 protein, alone or in combination.
In particular, the ten-n "hBD3 gene" refers to the full-length hBD3 nucleotide
sequence (e.g., contained in SEQ ID NO:11). However, it is also intended that
the term
encompass fragments of the hBD3 sequence, as well as other domains within the
full-length
hBD3 nucleotide sequence, as well as variants of hBD3. Furthermore, the terms
" hBD3
gene nucleotide sequence" or" hBD3 gene polynucleotide sequence" encompasses
DNA,
cDNA, and RNA (e.g., mRNA) sequences.
As used herein, the term "NIKS cells" refers to cells having the
characteristics of the
cells deposited as cell line ATCC CRL-12191.
The term "gene" refers to a nucleic acid (e.g., DNA) sequence that comprises
coding
sequences necessary for the production of a polypeptide or precursor (e.g.,
GKLF). The
polypeptide can be encoded by a full length coding sequence or by any portion
of the
coding sequence so long as the desired activity or functional properties
(e.g., enzymatic
activity, ligand binding, signal transduction, etc.) of the fall-length or
fragment are retained.
The term also encompasses the coding region of a structural gene and the
including
sequences located adjacent to the coding region on both the 5' and 3' ends for
a distance of
about 1 kb on either end such that the gene corresponds to the length of the
full-length
mRNA. The sequences that are located 5' of the coding region and which are
present on the
rnRNA are referred to as 5' untranslated sequences. The sequences that are
located 3' or
downstream of the coding region and that are present on the mRNA are referred
to as 3'
untranslated sequences. The term "gene" encompasses both cDNA and genomic
forms of a
gene. A genomic form or clone of a gene contains the coding region interrupted
with non-
coding sequences termed "introns" or "intervening regions" or "intervening
sequences."
Introits are segments of a gene that are transcribed into nuclear RNA (hnRNA);
introns may
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contain regulatory elements such as enhancers. 1ntrons are removed or "spliced
out" from
the nuclear or primary transcript; introns therefore are absent in the
messenger RNA
(mRNA) transcript. The mRNA functions during translation to specify the
sequence or
order of amino acids in a nascent polypeptide.
As used herein, the terms "nucleic acid molecule encoding," "DNA sequence
encoding," and "DNA encoding" refer to the order or sequence of
deoxyribonucleotides
along a strand of deoxyribonucleic acid. The order of these
deoxyribonucleotides
determines the order of amino acids along the polypeptide (protein) chain. The
DNA
sequence thus codes for the amino acid sequence.
As used herein, the term "recombinant DNA molecule" as used herein refers to a
DNA molecule that is comprised of segments of DNA joined together by means of
molecular biological techniques.
The term "isolated" when used in relation to a nucleic acid, as in "an
isolated
oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid
sequence that is
identified and separated from at least one contaminant nucleic acid with which
it is
ordinarily associated in its natural source. Isolated nucleic acid is present
in a form or
setting that is different from that in which it is found in nature. In
contrast, non-isolated
nucleic acids are nucleic _acids such as DNA and RNA found in the state they
exist in
nature. For example, a given DNA sequence (e.g., a gene) is found on the host
cell
=
chromosome in proximity to neighboring genes; RNA sequences, such as a
specific mRNA
sequence encoding a specific protein, are found in the cell as a mixture with
numerous other
mRNAs that encode a multitude of proteins, However, isolated nucleic acid
encoding KGF-
2 includes, by way of example, such nucleic acid in cells ordinarily
expressing KGF-2
where the nucleic acid is in a chromosomal location different from that of
natural cells, or is
otherwise flanked by a different nucleic acid sequence than that found in
nature. The
isolated nucleic acid, oligonucleotide, or polynucleotide may be present in
single-stranded
or double-stranded form. When an isolated nucleic acid, oligonucleotide or
polynucleotide
is to be utilized to express a protein, the oligonucleotide or polynucleotide
will contain at a
minimum the sense or coding strand (i.e., the oligonucleotide or
polynucleotide may be
single-stranded), but may contain both the sense and anti-sense strands (i.e.,
the
oligonucleotide or polynucleotide may be double-stranded).
As used herein the temi "portion" when in reference to a nucleotide sequence
(as in
"a portion of a given nucleotide sequence") refers to fragments of that
sequence. The
12
CA 02758178 2011-11-07
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fragments may range in size from four nucleotides to the entire nucleotide
sequence minus
one nucleotide (10 nucleotides, 20, 30, 40, 50, 100, 200, etc.).
As used herein the term "coding region" when used in reference to structural
gene
refers to the nucleotide sequences that encode the amino acids found in the
nascent
polypeptide as a result of translation of a mRNA molecule. The coding region
is bounded,
in eukaryotes, on the 5' side by the nucleotide triplet "ATG" that encodes the
initiator
methionine and on the 3' side by one of the three triplets that specify stop
codons (i.e., TAA,
TAG, TGA).
As used herein, the term "purified" or "to purify" refers to the removal of
contaminants from a sample.
As used herein, the term "vector" is used in reference to nucleic acid
molecules that
transfer DNA segment(s) from one cell to another. The term "vehicle" is
sometimes used
interchangeably with "vector."
The term "expression vector" as used herein refers to a recombinant DNA
molecule
containing a desired coding sequence and appropriate nucleic acid sequences
necessary for
the expression of the operably linked coding sequence in a particular host
organism.
Nucleic acid sequences necessary for expression in prokaryotes usually include
a promoter,
an operator (optional), and a ribosome binding site, often along with other
sequences.
Eukaryotic cells are known to utilize promoters, enhancers, and termination
and
polyadenylation signals.
A "regulatory sequence" refers to a polynucleotide sequence that is necessary
for
regulation of expression of a coding sequence to which the polynucleotide
sequence is
operably linked. The nature of such regulatory sequences differs depending
upon the host
organism. In prokaryotes, such regulatory sequences generally include, for
example, a
promoter, and/or a transcription termination sequence. In eukaryotes,
generally, such
regulatory sequences include, for example, a promoter and/or a transcription
termination
sequence. The term "regulatory sequence" may also include additional
components the
presence of which are advantageous, for example, a secretory leader sequence
for secretion
of the polypeptide attached thereto.
"Operably linked" refers to a juxtaposition wherein the components so
described are
in a relationship permitting them to function in their intended manner. A
regulatory
sequence is "operably linked" to a coding sequence when it is joined in such a
way that
13
CA 02758178 2011-11-07
WO 2005/012492 PCT/US2004/024627
expression of the coding sequence is achieved under conditions compatible with
the
regulatory sequence.
"PCR" refers to the techniques of the polymerase chain reaction as described
in
Saiki, et al., Nature 324:163 (1986); and Scharf et al., Science 233:1076-1078
(1986); U.S.
Pat. No. 4,683,195; and U.S. Pat. No. 4,683,202. As used herein, x is
"heterologous" with
respect to y if x is not naturally associated with y or x is not associated
with y in the same
manner as is found in nature.
By "pharmaceutically acceptable carrier," is meant any carrier that is used by
persons in the art for administration into a human that does not itself induce
any undesirable
side effects such as the production of antibodies, fever, etc. Suitable
carriers are typically
large, slowly metabolized macromolecules that can be a protein, a
polysaccharide, a
polylactic acid, a polyglycolic acid, a polymeric amino acid, amino acid
copolymers or an
inactive virus particle. Such carriers are well known to those of ordinary
skill in the art.
Preferably the carrier is thyroglobulin.
As used herein, the term "host cell" refers to any eukaryotic or prokaryotic
cell (e.g., bacterial cells such as E. coli, yeast cells, mammalian cells,
avian cells,
amphibian cells, plant cells, fish cells, and insect cells), whether located
in vitro or in vivo.
For example, host cells may be located in a transgenic animal.
The terms "overexpression" and "overexpressing" and grammatical equivalents,
are
used in reference to levels of mRNA to indicate a level of expression
approximately 3-fold
higher than that typically observed in a given tissue in a control or non-
transgenic animal.
Levels of mRNA are measured using any of a number of techniques !mown to those
skilled
in the art including, but not limited to Northern blot analysis. Appropriate
controls are
included on the Northern blot to control for differences in the amount of RNA
loaded from
each tissue analyzed (e.g., the amount of 28S rRNA, an abundant RNA transcript
present at
essentially the same amount in all tissues, present in each sample can be used
as a means of
normalizing or standardizing the KGF-2 mRNA-specific signal observed on
Northern
blots). The amount of mRNA present in the band corresponding in size to the
correctly
spliced KGF-2 transgene RNA is quantified; other minor species of RNA which
hybridize
to the transgene probe are not considered in the quantification of the
expression of the
transgenic mRNA.
The term "transfection" as used herein refers to the introduction of foreign
DNA into
eukaryotic cells. Transfection may be accomplished by a variety of means known
to the art
14
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including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated
transfection,
polybrene-mediated transfection, electroporation, microinjection, liposome
fusion,
lipofection, protoplast fusion, retroviral infection, and biolistics.
The term "stable transfection" or "stably transfected" refers to the
introduction and
integration of foreign DNA into the genome of the transfected cell. The term
"stable
transfectant" refers to a cell that has stably integrated foreign DNA into the
genomic DNA.
The term "transient transfection" or "transiently transfected" refers to the
introduction of foreign DNA into a cell where the foreign DNA does not
integrate into the
genome of the transfected cell. The foreign DNA persists in the nucleus of the
transfected
cell for several days. During this time the foreign DNA is subject to the
regulatory controls
that govern the expression of endogenous genes in the chromosomes. The term
"transient
transfectant" refers to cells that have taken up foreign DNA but have failed
to integrate this
DNA.
The term "calcium phosphate co-precipitation" refers to a technique for the
introduction of nucleic acids into a cell. The uptake of nucleic acids by
cells is enhanced
when the nucleic acid is presented as a calcium phosphate-nucleic acid co-
precipitate. The
original technique of Graham and van der Eb (Graham and van der Eb, Virol.,
52:456
[1973]) has been modified by several groups to optimize conditions for
particular types of
cells. The art is well aware of these numerous modifications.
The term "test compound" refers to any chemical entity, pharmaceutical, drug,
and
the like that can be used to treat or prevent a disease, illness, sickness, or
disorder of bodily
function, or otherwise alter the physiological or cellular status of a sample.
Test compounds
comprise both known and potential therapeutic compounds. A test compound can
be
determined to be therapeutic by screening using the screening methods of the
present
invention. A "known therapeutic compound" refers to a therapeutic compound
that has
been shown (e.g., through animal trials or prior experience with
administration to humans)
to be effective in such treatment or prevention.
The term "sample" as used herein is used in its broadest sense. A sample
suspected
of containing a human chromosome or sequences associated with a human clu-
omosorne
may comprise a cell, chromosomes isolated from a cell (e.g., a spread of
metaphase
chromosomes), genomic DNA (in solution or bound to a solid support such as for
Southern
blot analysis), RNA (in solution or bound to a solid support such as for
Northern blot
analysis), cDNA (in solution or bound to a solid support) and the like. A
sample suspected
CA 02758178 2015-07-08
of containing a protein may comprise a cell, a portion of a tissue, an.
extract containing one
Or more proteins and the like.
As used herein, the term "response", when used in reference to an assay,
refers to the =
generation of a detectable signal (e.g., accumulation of-reporter protein,
increase in ion
concentration, accumulation of a detectable chemical product).
As used herein, the teim "reporter gene" refers to a gene encoding a protein
that may
be assayed. Examples of reporter genes include, but are not limited to,
luciferase (See, e.g.,
deWet et al., Mol. Cell. Biol. 7:725 1.1987] and U.S. Pat Nos.,6,074,859;
5,976,796;
5,674,713; and 5,618,682 =green
fluorescent protein (e.g., GenBank Accession Number U43284; a number of GFP
variants
are commercially available from CLONTECH Laboratories, Palo Alto, CA),
chloramphenicol acetyltransferase, Beta-galactosidase, alkaline phosphatase,
and horse
radish peroxidase.
DETA_TLED DESCRIPTION
The present invention provides human skin equi-valents (e.g., NEE cells)
expressing
exogenous polypeptides (e.g., KGF-2 and antimicrobial polypeptides), and
compositions
and methods for making such cells. In addition, the present invention provides
methods for
treatment of wounds with such cells.
')0
I. Methods of Generating Host Cells
In. some embodiments, the present invention provides methods of generating
human
= tissues such as skin equivalents (e.g., from MKS cells) expressing
exogenous polypeptides
(e.g., KGF-2 and antimicrobial polypeptides).
=
A) Host Cells , =
Generally, any source of cells or cell line that can stratify into squamous
epithelia is
useful in the present invention. Accordingly, the present invention is not
limited to the -use
of any particular source of cells that are capable of differentiating into
squamous epithelia.
Indeed, the present invention contemplates the use of a variety of cell lines
and sources that
can differentiate into squarnous epithelia, including both primary and
immortalized
keratinocytes. Sources of cells include keratinocytes and dermal fibroblasts
biopsied from
humans and cavaderic donors (Auger et al., In Vitro Cell. Dev. Biol. ¨ Animal
36:96-103;
16 =
CA 02758178 2015-07-08
U.S. Pat. Nos. 5,968,546 and 5,693,332,
neonatal foreskins (A.sbill et al., Phalan. Research 17(9): 1092-97 (2000);
Meana et al.,
Burns 24:621-30 (1998); U.S. Pat. Nos. 4,485,096; 6,039,760; and 5,536,656,
and immortalized keratinocytes cell lines such as NIv11
cells (Baden, In Vitro Cell. Dev. Biol. 23(3).205-213 (1987)), PlaCaT cells
(Boucamp et al.,
J. cell. Boil. 106:761-771 (1988)); and NIKS cells (Cell line BC-1-Bp/SL; U.S.
Pat. No.
5,989,837, ATCC CRL-12191). Each of these cell lines
can be cultured or genetically modified as described below in order to produce
a cell line
capable of expressing an exogenous polypeptide.
In particularly preferred embodiments, MKS cells or cells derived from NIKS
cells
are utili7ed. NULLS cells (Cell line BC-1-Bp/SL; U.S. Pat Nos. 5,989,837,
6514,711, =
6,495,135, 6,485,724, and 6,214,567;
ATCC CRL-12191). The discovery of a novel human keratinocyte cell line (near-
diploid
immortalized keratinocytes or NIKS) provides an opportunity to genetiCally
engineer
humari keratinocytes for new therapeutic methods. A unique advantage of the
MKS cells is
that they are a consistent source of genetically-unifoiin, pathogen-free hi-
1'mm keratinocytes.
For this reason, they are useful for the application of genetic erineering and
genornic gene
expression approaches to provide skin equivalent cultures with properties more
similar-to
human skin. Such systems will provide an important alternative to the use of
animals for
testing compounds and foiiiiulations. The NIKS keratinocyte cell line,
identified and
characterized at the University of Wisconsin, is nontumorigenic, exhibits a
stable karyotype,
and undergoes normal differentiation both in monolayer and organotypic
culture. NIKS
cells form fully stratified skin equivalents in culture. These cultures are
indistinguishable
by all criteria tested thus far from organotypic cultures foithed fi-om
primary human
keratinocytes. Unlike primary cells however, the. immortalized NIKS cells will
continue to
proliferate in inonolayer culture indefinitely. This provides an opportunity
to genetically
manipulate the cells and isolate new clones of cells with new useful
properties (Allen-
Hoffmann et al., J. Invest. Dermatol., 114(3): 444-455 (2000)).
The MKS cells arose from the BC-1-Ep strain of human neonatal foreskin
keratinocytes isolated from an apparently noinial male infant. In early
passages, the BC-1-
Ep cells exhibited no morphological or growth characteristics that were
atypical for cultured
normal Imman keratinocytes. Cultivated BC-1-Ep cells exhibited stratification
as well as
features of programmed cell death. To determine repheative lifespan, the BC-1-
Ep cells
were serially cultivated to senescence in standard keratinocyte growth medium
at a density
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CA 02758178 2011-11-07
WO 2005/012492 PCT/US2004/024627
of 3 x 1O cellsper 100-mm dish and passaged at weekly intervals (approximately
a 1:25
split). By passage 15, most keratinocytes in the population appeared senescent
as judged by
the presence of numerous abortive colonies that exhibited large, flat cells.
However, at
passage 16, keratinocytes exhibiting a small cell size were evident. By
passage 17, only the
small-sized keratinocytes were present in the culture and no large, senescent
keratinocytes
were evident. The resulting population of small keratinocytes that survived
this putative
crisis period appeared morphologically uniform and produced colonies of
keratinocytes
exhibiting typical keratinocyte characteristics including cell-cell adhesion
and apparent
squame production. The keratinocytes that survived senescence were serially
cultivated at a
density of 3 x 105 cells per 100-mm dish. Typically the cultures reached a
cell density of
approximately 8 x 106 cells within 7 days. This stable rate of cell growth was
maintained
through at least 59 passages, demonstrating that the cells had achieved
immortality. The
keratinocytes that emerged from the original senescencing population were
originally
designated BC-1-Ep/Spontaneous Line and are now termed NIKS. The NIKS cell
line has
been screened for the presence of proviral DNA sequences for HIV-1, HIV-2,
EBV, CMV,
HTLV-1, HTLV-2, HBV, HCV, B-19 parvovirus, HPV-16 and HPV-31 using either PCR
or Southern analysis. None of these viruses were detected.
Chromosomal analysis was performed on the parental BC-1-Ep cells at passage 3
and NIKS cells at passages 31 and 54. The parental BC-1-Ep cells have a normal
chromosomal complement of 46, XI'. At passage 31, all NIKS cells contained 47
chromosomes with an extra isochromosome of the long arm of chromosome 8. No
other
gross chromosomal abnormalities or marker chromosomes were detected. At
passage 54,
all cells contained the isochromosome 8.
The DNA fingerprints for the NIKS cell line and the BC-1-Ep keratinocytes are
identical at all twelve loci analyzed demonstrating that the NIKS cells arose
from the
parental BC-1-Ep population. The odds of the NIKS cell line having the
parental BC-1-Ep
DNA fingerprint by random chance is 4 x 1046. The DNA fingerprints from three
different
sources of human keratinocytes, ED-1-Ep, SCC4 and SCC13y are different from
the BC-1-
Ep pattern. This data also shows that keratinocytes isolated from other
humans, ED-1-Ep,
SCC4, and SCC13y, are unrelated to the BC-1-Ep cells or each other. The NIKS
DNA
fingerprint data provides an unequivocal way to identify the NIKS cell line.
Loss of p53 function is associated with an enhanced proliferative potential
and
increased frequency of immortality in cultured cells. The sequence of p53 in
the N1KS cells
is identical to published p53 sequences (GenBank accession number: M14695). In
humans,
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CA 02758178 2011-11-07
WO 2005/012492 PCT/US2004/024627
p53 exists in two predominant polymorphic forms distinguished by the amino
acid at codon
72. Both alleles of p53 in the NIKS cells are wild-type and have the sequence
CGC at
codon 72, which codes for an arginine. The other common form of p53 has a
proline at this
position. The entire sequence of p53 in the NIKS cells is identical to the BC-
1-Ep
progenitor cells. Rb was also found to be wild-type in MKS cells.
Anchorage-independent growth is highly correlated to tumorigenicity in vivo.
For
this reason, the anchorage-independent growth characteristics of NIKS cells in
agar or
methylcellulose-containing medium was investigated. After 4 weeks in either
agar- or
methylcellulose-containing medium, NIKS cells remained as single cells. The
assays were
continued for a total of 8 weeks to detect slow growing variants of the NIKS
cells. None
were observed.
To determine the tumorigenicity of the parental BC-1-Ep keratinocytes and the
immortal NIKS keratinocyte cell line, cells were injected into the flanks of
athymic nude
mice. The human squamous cell carcinoma cell line, SCC4, was used as a
positive control
for tumor production in these animals. The injection of samples was designed
such that
animals received SCC4 cells in one flank and either the parental BC-1-Ep
keratinocytes or
the NIKS cells in the opposite flank. This injection strategy eliminated
animal to animal
variation in tumor production and confirmed that the mice would support
vigorous growth
of tumorigenic cells. Neither the parental BC-1-Ep keratinocytes (passage 6)
nor the NIKS
keratinocytes (passage 35) produced tumors in athymic nude mice.
N1KS cells were analyzed for the ability to undergo differentiation in both
surface
culture and organotypic culture. For cells in surface culture, a marker of
squamous
differentiation, the formation cornified envelopes was monitored. In cultured
human
keratinocytes, early stages of cornified envelope assembly result in the
formation of an
immature structure composed of involucrin, cystatin-a and other proteins,
which represent
the inneimost third of the mature cornified envelope. Less than 2% of the
keratinocytes
from the adherent BC-1-Ep cells or the 1\11KS cell line produce cornified
envelopes. This
finding is consistent with previous studies demonstrating that actively
growing,
subconfluent keratinocytes produce less than 5% cornified envelopes. To
determine
whether the MKS cell line is capable of producing cornified envelopes when
induced to
differentiate, the cells were removed from surface culture and suspended for
24 hours in
medium made semi-solid with methylcellulose. Many aspects of tenninal
differentiation,
including differential expression of keratins and comified envelope fonnation
can be
triggered in vitro by loss of keratinocyte cell-cell and cell-substratum
adhesion. The NIKS
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WO 2005/012492 PCT/US2004/024627
keratinocytes produced as many as and usually more comified envelopes than the
parental
keratinocytes. These findings demonstrate that the NIKS keratinocytes are not
defective in
their ability to initiate the formation of this cell type-specific
differentiation structure.
To confirm that the NIKS keratinocytes can undergo squamous differentiation,
the
cells were cultivated in organotypic culture. Keratinocyte cultures grown on
plastic
substrata and submerged in medium replicate but exhibit limited
differentiation.
Specifically, human keratinocytes become confluent and undergo limited
stratification
producing a sheet consisting of 3 or more layers of keratinocytes. By light
and electron
microscopy there are striking differences between the architecture of the
multilayered sheets
formed in tissue culture and intact human skin. In contrast, organotypic
culturing
techniques allow for keratinocyte growth and differentiation under in vivo-
like conditions.
Specifically, the cells adhere to a physiological substratum consisting of
dermal fibroblasts
embedded within a fibrillar collagen base. The organotypic culture is
maintained at the air-
medium interface. In this way, cells in the upper sheets are air-exposed while
the
proliferating basal cells remain closest to the gradient of nutrients provided
by diffusion
through the collagen gel. Under these conditions, coma tissue architecture is
formed.
Several characteristics of a normal differentiating epidermis are evident. In
both the
parental cells and the NIKS cell line a single layer of cuboidal basal cells
rests at the
junction of the epidermis and the dermal equivalent. The rounded morphology
and high
nuclear to cytoplasmic ratio is indicative of an actively dividing population
of keratinocytes.
In normal human epidermis, as the basal cells divide they give rise to
daughter cells that
migrate upwards into the differentiating layers of the tissue. The daughter
cells increase in
size and become flattened and squamous. Eventually these cells enucleate and
form
cornified, keratinized structures. This normal differentiation process is
evident in the upper
layers of both the parental cells and the NIKS cells. The appearance of
flattened squamous
cells is evident in the upper layers of keratinocytes and demonstrates that
stratification has
occurred in the organotypic cultures. In the uppermost part of the organotypic
cultures the
enucleated squames peel off the top of the culture. To date, no histological
differences in
differentiation at the light microscope level between the parental
keratinocytes and the
MKS keratinocyte cell line grown in organotypic culture have been observed.
To observe more detailed characteristics of the parental (passage 5) and NIKS
(passage 38) organotypic cultures and to confirm the histological
observations, samples
were analyzed using electron microscopy. Parental cells and the irrunortalized
human
keratinocyte cell line, NIKS, were harvested after 15 days in organotypic
culture and
CA 02758178 2015-07-08
sectioned perpendicular to the basal layer to show the extent of
stratification. Both the
parental cells and the I\T.KS cell line undergo extensive stratification in
organotypic culture
and form structures that are characteristic of nointal human epidermis.
Abundant
desmc.)somes are formed in organoty-pic cultures of parental cells and the
NIKS cell line.
The formation of a basal larnin.a and associated hemidesmosomes in the basal
kcratinocyte
layers of both the parental cells and the cell line was also noted.
Hemidesmosomes are specialized structures that increase adhesion of the
keratinocytes to the basal lamina and help maintain the integrity and strength
of the tissue.
The presence of these structures was especially evident in areas where the
parental cells or
the NIKE cells had attached directly to the porous support. These frndings are
consistent
with earlier ultrastructural findings using human foreskin keratinocytes
cultured on a
fibroblast-containing porous support. Analysis at both the light and electron
microscopic
levels demonstrate that the NEE cell line in organotypic culture can stratify,
differentiate,
and fon-ar structures such as desnaosomes, basal lamina, and hemidesmosomes
found in
normal human epidermis.
B) KGF-2
In some embodiments, the present invention provides human skin equivalents
(e.g.,
keratinocytes) that express exogenous KGF-2 protein. KGF-2 is a 208 amino acid
protein
that influences normal keratinocyte and epithelial cells to proliferate and
migrate to wound
sites. Protein and nucleic acid sequences for KGF-2 are. provided in 11.5.
Pat, No.
6,077,692,,
KGF-2 promotes wound healing in tissues containing keratinocytes and
fibroblasts
by having a positive proliferative effect on epithelial cells and mediating
keratinoc;yte
migration. In addition, KGF-2 promotes wound healing by increasing deposition
of
granulation tissue and collagen, and maturation of collagen (Soler et al.,
Wound Repair
Regen. 7(3):172-178 (1999)).
= C) Antimicrobial polypeptides
In SOMe embodiments, the present invention provides human skin equivalents
(e.g.,
= keratinocytes) that express exogenous antimicrobial polypeptides. In
intact human skin, the
stratum comeum serves as the first line of defense against microbial
organisms. The
stratum comeum is the uppermost, nonviable, desiccated layer of the epidermis
that is
composed of fully differentiated keratinocytes. The innate immune response
prevents
21
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CA 02758178 2011-11-07
WO 2005/012492 PCT/US2004/024627
invasion of microbial organisms if the outer most layer of the skin barrier is
penetrated. This
response includes phagocytosis by macrophages and neutrophils and their
production of
reactive oxygen intermediates that kill microbial agents. Associated with this
line of
defense are antimicrobial peptides that are naturally expressed and localized
to the upper
layers of the epidermis. The most thoroughly studied human antimicrobial
peptides belong
to two subfamilies, the a- and 13-defensins, which differ from one another by
their disulfide
bond pairing, genomic organization and tissue distributions (Ganz, T. and J.
Weiss, Semin
Hernatol, 1997. 34(4): p. 343-54). The P-defensins are characteristically
found in epithelial
tissues and are expressed in human keratinocytes. This defensin subfamily
demonstrates
strong antimicrobial activity against a broad spectrum of pathogenic agents,
including
bacteria, fungi and viruses.
Microorganisms have difficulty acquiring resistance to the defensin peptides,
making these peptides very attractive for therapeutic use as antibiotics
(Schroder, J.M.,
Biochem Pharrnacol, 1999. 57(2): p. 121-34). In clinical trials, defensin
peptides applied to
skin have been found to be safe (Hancock, R.E., Lancet, 1997. 349(9049): p.
418-22). The
safety of topically-applied defensins is consistent with the finding that
human epidermal
keratinocytes express defensin peptides in vivo.
In the human genome, all known defensin genes cluster to a < 1 Mb region of
chromosome 8p22-p23; these findings suggest an evolutionary conservation of
this gene
family. Harder, J., et al., Mapping of the gene encoding hunzan beta-defensin-
2 (DEFB2) to
chronzosonze region 8p22-p23.1. Genomics, 1997. 46(3): p, 472-5. It is
generally accepted
that evolutionarily conserved genes maintain some overlap in gene function.
The defensin
gene family is no exception to this theory. The defensin genes encode small (3-
51cDa),
cationic molecules characterized by an amphipathic structure and have six
cysteine residues
that form three intramolecular disulfide bonds (see Figure 11). These cationic
regions are
thought to be attractive to the anionic surfaces of most bacteria. The human
defensin gene
family is divided into two subfamilies: the a-defensins and p-defensins that
differ from one
another by their disulfide bond pairing, genomic organization and tissue
distributions. The
a- and P-defensins share similarity in tertiary structure and both contain
triple stranded
antiparallel beta sheets (Pardi, A., et al., Bochemistry, 1992. 31(46): p.
11357-64;
Zimmermann, G.R., et al., Biochemistry, 1995. 34(41): p. 13663-71). However,
their
antimicrobial mechanisms of action are distinct from one another.
Historically the a-defensins have been found in storage granules of
specialized cell
types such as neutrophils and Paneth cells of the small intestine, whereas the
P-defensins are
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WO 2005/012492
PCT/US2004/024627
expressed in epithelial tissues. The a-defensins also have an inhibitory pro-
region in their
amino-terminal sequence, which is cleaved off after release from granules. The
pro-region
is likely to contain a granule targeting motif but may function independently
as a protease
inhibitor. The broad spectrum of antimicrobial activity is mediated in part by
permeabilization of biological membranes. Although extremely potent for
killing invading
microorganisms, a-defensins have also been shown to be toxic to eukaryotic
cell types
(Lichtenstein, A., et al., Blood, 1986. 68(6): p. 1407-10; Olcrent et al., Am
Rev Respir Dis,
1990. 141(1): p. 179-85). The a-defensin-induced pleiotropic cell killing
activity makes
this subfamily of defensins unattractive as a gene candidate for expression in
living human
skin substitutes.
Keratinocytes of the skin and other epithelia harbor endogenously expressed
members of the f3-defensins. To date, there have been six distinct genes
identified. Three
of these human P-defensin genes, hBD-1, & hBD-3, are expressed in epidermal
keratinocytes of the skin. The first exon encodes the signal sequence and
propeptide and
the second exon encodes the mature peptide. Amino acid sequence alignment
highlighting
conserved residues and the characteristic six cysteine residues of the human
f3-defensins 1-3
are shown in Figure 10. The disulfide covalent bonds required for secondary
structure of
the active peptide are demonstrated in Figure 11.
Several factors are thought to contribute to the antimicrobial action of the P-
defensins on microbes. First because of their cationic and amphiphilic
characteristics,
antimicrobial peptides bind and insert into the cytoplasmic membrane, where
they assemble
into multinieric pores, and destroy the target microbe by changing membrane
conductance
and altering intracellular function (White, S.H., W.C. Wimley, and M.E.
Selsted, Curr Opin
Struct Biol, 1995. 5(4): p. 521-7; Boman, H.G., Annu Rev Immunol, 1995. 13: p.
61-92).
Most antimicrobial peptides kill microorganisms by forming pores in the cell
membrane.
These peptides are not toxic to mammalian cells due to the sensitivity of
these peptide
antibiotics to cholesterol and phospholipids, major components of mammalian
cell
membranes. The f3-defensins are attractive candidates for therapeutic use as
antibiotics
since it is difficult for microorganisms to acquire resistance to the
peptides' bactericidal
mechanism of action (Schroder, J.M., Biochem Phannacol, 1999. 57(2): p. 121-
34).
When expressed, the 13-defensin peptides appear to initially localize to the
cytoplasm
of undifferentiated or less differentiated keratinocytes. As these cells
differentiate and
move closer to the epidemial surface, they secrete these antimicrobial
peptides onto the
keratinocyte membrane or into the intracellular space. The signal peptide
sequence is
23
CA 02758178 2011-11-07
WO 2005/012492 PCT/US2004/024627
thought to contribute to the specialized localization of this active peptide.
Finally human (3-
defensin peptides accumulate in the dehydrated cells of the epidermal surface.
Studies
demonstrate that, although the three 0-defensin genes are very similar, their
expression is
determined by completely different regulatory mechanisms (Frye, M., J. Bargon,
and R.
Gropp, J Mol Med, 2001. 79(5-6): p. 275-82).
The burn wound is an ideal environment for bacterial growth and provides a
pathway for microbial invasion. Luterman and coworkers concluded "Burned skin
is a
nidus and portal for bacterial invasion, causing burn wound sepsis, the
leading cause of
death in burn units around the world" (Luterman, A., C.C. Dacso, and P.W.
Curreri, Am J
Med, 1986. 81(1A): p. 45-52). Infection is further promoted by skin loss and
post burn
inffnuno-suppression. As expected, human defensin gene expression is
diminished in full
thickness bum wounds most probably due to the destruction of the epithelium.
For
example, human fl-defensin gene (1)BD-2) expression is virtually undetectable
in the burn
wound suggesting the loss of defensins due to thermal destruction of the skin
(Milner, S.M.
and M.R. Ortega, Bums, 1999. 25(5): p. 411-3). A routinely used debridement
procedure
may also contribute to significant removal of epithelia in a wound bed.
Debridement speeds
the healing of ulcers, burns, and other wounds by removing dead tissue so that
the
remaining living tissue can adequately heal. Wounds that contain non-living
(necrotic)
tissue take longer to heal because necrotic debris is a nutrient source for
bacteria in a
wound. The debridement procedure introduces a potential risk that surface
bacteria may be
introduced deeper into the body, causing infection.
Bacteria typically encountered in a burn wound include E. co/i, P. aeruginosa,
S.
aureus, and C. albicans (Ileggers, J.P., Treatment of iy'ection in burns, H.
DN, Editor.
1996, 'WB Saunders: London. p. 98-135). All of these microbes are killed by
one or more
of the 13-defensin antimicrobial peptides.
Some 13-defensin family members are upregulated in response to inflammatory
stimuli or bacterial invasion. Others remain non-responsive, downregulated or
suppressed
in response to inflammatory stimuli or bacterial exposure. In unwounded,
intact skin, the
calculated epidemial concentrations of13-defensin peptides are well within the
range needed
for their antimicrobial effects. The 13-defensins possess chemotactic activity
for immature
dendritic cells and memory T cells. These chemotactic responses require much
lower
concentrations than required for antimicrobial activity (Yang, D., et al.,
Science, 1999.
286(5439): p. 525-8). As a result of this cross-talk, the P-defensins are
thought to mediate
an important link between innate and adaptive immunity. Therefore, the P-
defensins appear
24
CA 02758178 2011-11-07
WO 2005/012492 PCT/US2004/02.1627
to play a multifunctional role by promoting both an adaptive immune response
and
inflammation, while facilitating wound healing through their antimicrobial
activity.
Adaptive immunity is promoted through the endogenous antimicrobial peptides in
healthy
human skin and likely provides an effective shield from microbial infection;
however,
patients with unhealthy or chronic skin wounds would also benefit from boosted
local
antimicrobial peptide levels.
The liBD-1 gene encodes for a 3.9 kDa basic peptide that was originally
identified
in hemofiltrates from human patients with end stage renal disease (Bensch,
K.W., et al.,
FEBS Lett, 1995. 368(2): p. 331-5). hBD-1 bactericidal activity is
predominantly against
grant negative bacteria such as E. co/i and P. aeruginosa. Constitutive hBD-1
expression
has been observed in skin from various sites on the body. The overexpression
of hBD-1 in
immortalized human skin cells (HaCat) is associated with keratinocyte cell
differentiation.
Overexpression was confirmed to have no effect on proliferating cells. The
present
invention is not limited to a particular mechanism. Indeed, an understanding
of the
mechanism is not necessary to practice the present invention. Nonetheless, it
is
contemplated that P-defensin gene expression is a consequence of
differentiation, rather
than an inducer of differentiation in keratinocytes (Frye, M., J. Bargon, and
R. Gropp, J Mol
Med, 2001. 79(5-6): p. 275-82). hBD-1 expression in differentiated
keratinocyte cells is
inhibited upon exposure to bacteria. The present invention is not limited to a
particular
mechanism. Indeed, an understanding of the mechanism is not necessary to
practice the
present invention. Nonetheless, it is contemplated that this result indicates
that this factor is
an important mediator of the healing process in regenerating epithelia. These
studies
confirm the upregulation of hBD-1 expression is a result of factors not
associated with an
inflammatory response. This antimicrobial peptide is not induced by
inflammatory
cytokines, which is consistent with the lack of cytokine-responsive
transcription factor
regulatory elements in the hBD-1 5 'regulatory sequences.
hBD-2 peptide was originally identified in desquamated squames of psoriatic
skin
and hBD-2 gene expression has since been identified in normal human
keratinocytes
(Harder, J., et al., Genomics, 1997. 46(3): p. 472-5). This gene encodes for a
4 kDa basic
peptide. Variable endogenous levels of expression have been observed when
comparing
skin from various sites on the body, with the most prominent expression
observed in facial
skin and foreskin. Expression is localized to the suprabasal layers and the
stratum corneum
of intact skin. Low levels of hBD-2 protein have been detected in the
cytoplasm of
keratinocytes in basal layers of skin tissue. These proteins are believed to
be secreted into
CA 02758178 2011-11-07
WO 2005/012492 PCT/US2004/024627
the cell membrane or intercellular spaces as the cells achieve a suprabasal
position in the
tissue and eventually concentrate in the dehydrated cells of the stratum
comeum. hBD-2
peptide efficiently combats clinical isolates of gram negative bacteria such
as P. aeruginosa
and E. coli, while only having a bacteriostatic effect, at high
concentrations, on gram
positive bacterial strains such as S. aureus (Liu, A.Y., et al., J Invest
Dermatol, 2002.
118(2): p. 275-81). Studies show that endogenous expression is triggered by
inflammatory
cytoldnes as well as exposure to bacteria. Finally, not only does hBD-2 have
antimicrobial
activity, it also modulates the inflammatory response in various skin
conditions (Garcia,
J.R., et al., Cell Tissue Res, 2001. 306(2): p. 257-64).
The hBD-3 gene encodes for a 5 kDa basic peptide that was identified by
screening
genomic sequences for antimicrobial activity and the ability to activate
monocytes. The
gene was cloned from differentiated respiratory epithelial cells. Strongest
expression has
been exhibited in the skin and tonsil. Endogenous expression is triggered by
inflammation,
and therefore, hBD-3 is not constitutive but rather a readily inducible
antimicrobial peptide.
This peptide is also a potent chemoattractant for monocytes and neutrophils,
which are
strongly involved in the innate immune response (Garcia, J.R., et al., Cell
Tissue Res, 2001.
306(2): p. 257-64). hBD-3 possesses a broad spectrum antimicrobial peptide
activity at low
micromolar concentrations, against many potential pathogenic microbes
including P.
aeruginosa, S. pyrogenes, multiresistant S. aureus, vancomycin-resistant E.
faecium, and the
yeast C. albicans. liBD-3 gene expression is also induced in HaCat and
cultured skin-
derived keratinocytes when stimulated with heat-inactivated bacteria (Harder,
J., et al.,
Nature, 1997. 387(6636): p. 861). It is speculated that some disorders of
defective innate
immunity, such as unexplained recurrent infections of particular organs, may
be caused by
abnormalities that reduce expression of one or more genes that encode
defensins or other
antimicrobial peptides. Synthetic 1BD-3 protein exhibits a strong
antimicrobial activity
against gram-negative and gram-positive bacteria and fungi.
The present invention contemplates that the overexpression of exogenous
antimicrobial polypeptides in human skin equivalents speeds wound healing and
prevents
infection of the wound. In some preferred embodiments, the antimicrobial
polypeptide is
overexpressed in the human skin equivalent is human beta defensins 1, 2, or 3
or
combinations thereof.
The present invention is not limited to the expression of any particular
exogenous
antimicrobial polypeptide in the human skin equivalents. Indeed, the
expression of a variety
of antimicrobial polypeptides is contemplated, including, but not limited to
the following:
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CA 02758178 2011-11-07
WO 2005/012492 PCT/US2004/024627
following: magainin (e.g., magainin I, magainin 11, xenopsin, xenopsin
precursor fragment,
caerulein precursor fragment), magainin I and Tf analogs (PGLa, magainin A,
magainin G,
pexiganin, Z-12, pexigainin acetate, D35, MST-78A, MGO [K10E, Kl1E, F12W-
magainin
2], MG2+ [K10E, Fl 2W-magainin-2], MG4+ [F12W-magainin 2], MG6+ [fl2W, E1 9Q-
magainin 2 amide], MSI-238, reversed magainin If analogs [e.g., 53D, 87-ISM,
and A87-
ISM], Ala-magainin 11 amide, magainin II amide), cecropin P1, cecropin A,
cecropin B,
indolicidin, nisin, ranalexin, lactoferricin 13, poly-L-lysine, cecropin A (1-
8)-magainin II (1-
12), cecropin A (1-8)-melittin (1-12), CA(1-13)-MA(1-13), CA(1-13)-ME(1-13),
gramicidin, gramicidin A, gramicidin D, gramicidin S, alamethicin, protegrin,
histatin,
dermaseptin, lentivirus arnphipathic peptide or analog, parasin I, lycotoxin I
or IL
globomycin, gramicidin S, surfactin, ralinomycin, valinomycin, polymyxin B,
PM2 [ (+/-)
1-(4-aminobuty1)-6-benzylindane], PM2c [ (+/-) -6-benzy1-1-(3-
carboxypropypindane],
PM3 [(+/-)1-benzyl-6-(4-arninobutyl)indane], tachyplesin, buforin I or II,
misgurin,
melittin, PR-39, PR-26, 9-phenylnonylamine, (KLAKKLA)n, (KLAKLAK)n, where n =
1,
2, or 3, (KALKALK)3, KLGKKLG)n, and KAAKKAA)n, wherein N = 1, 2, or 3,
paradaxin, Bac 5, Bac 7, ceratoxin, mdelin 1 and 5, bombin-like peptides, PGQ,
cathelicidin, HD-5, Oabac5alpha, ChBac5, SMAP-29, Bac7.5, lactoferrin,
granulysin,
thionin, hevein and knottin-like peptides, MPG1, lbAMP, snakin, lipid transfer
proteins,
and plant defensins. Exemplary sequences for the above compounds are provided
in Table
1. In some embodiments, the antimicrobial peptides are synthesized from L-
amino acids,
while in other embodiments, the peptides are synthesized from or comprise D-
amino acids.
In some preferred embodiments of the present invention, the antimicrobial
polypeptide is a defensin. In certain embodiments, the defensin comprises the
following
consensus sequence: (SEQ ID NO:107 - XCNICRN2CN3ERN4CN5GN6CCX2, wherein N
and X represent conservatively or nonconservatively substituted amino acids
and N1 = 1, N2
= 3 or 4, N3 = 3 or 4, N4 = 1, 2, or 3, N6 = 5-9, X1 and X2 may be present,
absent, or equal
from 1-2.
In certain embodiments, mutant defensins are utilized in the methods and
= compositions of the present invention. For example, in some embodiments,
disulfide bond
fomiation in beta-defensin 3 is disrupted by mutation of one or more cysteine
residues. In
= preferred embodiments, 5 of the 6 cysteine residues (e.g., Cys40, Cys45,
Cys55, Cys62, and
Cys63) are mutated to alanine or other uncharged amino acid not capable of
forming
disulfide bonds. The present invention is not limited to a particular
mechanism. Indeed, an
understanding of the mechanism is not necessary to practice the present
invention.
27
CA 02758178 2011-11-07
WO 2005/012492 PCT/US2004/024627
Nonetheless, it is contemplated that disruption of disulfide bond formation in
beta-defensin
3 increases the antimicrobial activity of the protein (See e.g., Hoover et
al., Antimicrobial
agent and chemotherapy 47:2804 (2003) and Wu et al., PNAS 100:8880 (2003)).
The hBD-
3 mutants of the present invention may have altered (e.g., greater or less)
antimicrobial
activity than wild type hBD-3 or they may have similar antimicrobial activity.
It is further
contemplated that the disruption of disulfide bonds reduces or eliminates the
ability of h13D-
3 to elicit a chemotactic response. The elimination of chemotactic response
may be
desirable for avoidance of immune response to skin equilavents grafted onto
hosts (e.g.,
human hosts).
In other embodiments, glycine to alanine substitutions are generated in hBD-3
(e.g.,
Gly38A1a). In some embodiments, the both Gly-Ala and Cys-Ala substitutions are
generated in the same hBD-3 polypeptide.
In some embodiments, antimicrobial polypeptides are modified to include a
secretion signal peptide at the N-terniinus of the antimicrobial peptides to
create a chimeric
(hybrid) protein. It is contemplated that such signal sequences allow for the
free secretion
of antimicrobial peptides, rather than facilitating their association with the
cell surface. The
antimicrobial peptides have an endogenous signal secretion peptide that
directs the
immature peptide to the golgi apparatus and eventual secretion into
intracellular spaces.
These peptides appear to be tightly associated with the cell surfaces, and not
"freely"
secreted. In some embodiments, the IL-2 Signal secretion peptide is used (CTT
GCA CTT
GTC ACA AAC AGT GCA CCT; SEQ ID NO:108).
In other embodiments, the antimicrobial polypeptide is a human cathelicidin
(hCAP18) polypeptide (SEQ ED NO:47).
The present invention is not limited to any particular antimicrobial peptide.
Indeed,
media comprising a variety of antimicrobial polypeptides are contemplated.
Representative
antimicrobial polypeptides are provided in Table 1 below.
Table 1
Antimicrobial Peptides
SEQ ID Name Organism Sequence
NO:
13 beta-defensin 1 MRTSI'LLLFTLCLLLSEMASGGNFLTGLGHR
SDHYNCVSSGGQCLYSACPIFTKIQGTCYRG
KAKCCK
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Table 1
Antimicrobial Peptides
SEQ ID Name Organism Sequence
NO:
14 beta-defensin 2 Human MRVLYLLFSFLFIFLMPLPGVFGGIGDPVTCL
KSGAICHP'VFCPRRYKQIGTCGLPGTKCCKK
15 beta-defensin 3 Hunzan MREHYLLFALLELFLVPVPGI-IGGIINTLQKYY
CRVRGGRCAVLSCLPKEEQIGKCSTRGRKCC
RRKK
16 lingual antimicrobial Bos taunts
mrlhhillallflvlsagsgftqgvrrisqscrmkgicvp
peptide precursor ircpgsmrqigtclgaqvkccnk
(Magainin)
17 antimicrobial peptide Xenopus laevis
GvIsnvigylIcklgtgalnavIkq
PGQ
18 Xenopsin Xenopus laevis
mykgifIcvllavicanslatpssdadedndeveryvrgw
aslcigqtlgkialcvglkeliqpIcreamIrsaeaulapwil
19 magainin precursor Xenopus laevis
nifkglficsliavicanalpqpeasadedmderevrgigk
flbsagkfgkafvgeinikskrdaeavgpeafadedldere
vrgigkflhsakkfgkafvgeirrmskrdaeavgpeafade
dlderevrgigkflhsaktfgkafvgeimnslcrdaeavgp
eafadedlderevrgigIcfllisakkfgkafvgeinmslcrd
aeavgpeafadedfderevrgigkflhsalddgkafvgei
mnslcrdaeavgpeafadedlderevrgigIcflhsalckfgk
afvgeirrmslcrdaeavddrrwve
20 tachyplesin I Tachypleus k-wcfrveyrgicyncr
gigas
21 tachyplesin 11 Tachypleus rwcfrvcyrgicyrker
gigas
22 buforin I Bufo bufo
msgrgkqggkvrakaktrssraglqfpvgrvhrllrkgny
gagarizans
aqrvgagapvylaavleyltaeilelagnaardnklctrii
prh1q1avrndeehildlggvtiaqggv1pniqavllpkt
esskpaksk
23 buforin 11 Bufo bufo trssraglqfpvgrvhrllrk
gagarizans
24 cecropin A Bombyx mori
mnfvrilsfvfalvlaIgavsaapeprwklfkkiekvgrn
wdglikagpaiavigqakslgk
25 cecropin B BOMbyX Mori
mnfakilsfvfalvIalsmtsaapepnvkifiddelangrn
irdgivkagpaievlgsakaigk
26 cecropin C Drosophila
rnnfykifvfvalilaisigqseagwlkIclgkrierigqht
melanogaster rdatiqglgiaqqaanvaatarg
27 cecropin PI Sus scrofa swIsktaldclensaldcrisegiaiaiqggpr
28 Indolicidin Bos taurus ilpwkwpwwpwrr
29 Nisin Lactococcus itsisletpgclagalmgcnmktatcbcsibvsk
lactis
29
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PCT/U52004/024627
Table 1
Antimicrobial Peptides
SEQ ID Name Organism Sequence
NO:
30 Ranalexin Rana flgglikivparnicavtklcc
catesbeiana
31 lactoferricin B Bos taurus fkerrwqwrmIcIdgapsitcwraf
32 Protegrin-1 Sus scrofa rggrlcycrrrfcvcvgrx
33 Protegrin-2 Sus scrofa ggrIcycrrrfcicvg
34 histatin precursor H01710 sapiens
mkffvfalilalmIsmtgadshalcrhhgykTkihelchhsh
rgyrsnylydn
35 histatin 1 Macaca dsheerhhgrhghhicygrldhekiihshrgyrsnylydn
fascicularis
36 derniaseptin Phyllomedusa alwictrnlIcklgtmalhagkaalgaaadtisqtq
sauvagei
37 dermaseptin 2 Phyllomedusa alwftmlkIdgtinalliagkaalgaaantisqgtq
sauvagei
38 dermaseptin 3 Phyllomedusa alwicnni1kgigklagkaalgavidclvgaes
sauvagei
39 Misgurin Misgurnus rqrveelskfskIcgaaaark
anguillicaudatu
40 Melittin Apis mellifera gigavlicvittglpaliswisrkkrqq
41 pardaxin-1 Pardachirus gffaliplciisspffictIlsavgsalsssgeqe
pavoninus
42 pardaxin-2 Pardachirus = gffalipkiisspifktIlsavgsalsssggqe
pavoninus
43 Bactenecin 5 precursor Bos taurus
metqraslslgrcslw1111g1v1psasaqalsyreavlr
avdqthersseanlyrIleldptpriddldpgrrkpvsfrv
ketdcprtsqqpIeqedfkenglvkqcvgtvtldpsndqf
dincnelqsvrfrppirrppirppfyppfrppirppifpp
irPPfrPP1gPfPgrr
44 bac tenecin precursor Bos taurus
metpraslsIgrwslwIllIglalpsasaqalsyreavIr
avdqlneqssepthyrlIeldcippqddedpdsplcrvsfrv
ketvcsrttqqppeqcdfkengllIcrcegtvtldqvrgnf
ditcnnhqsiritkqpwappqaarlcrivvirvcr
45 ceratotoxin A Ceratitis sigsalkkalpvalckigkialpiakaalp
capitata
46 ceratotoxin B Ceratitis sigsafkkalpvakkigkaalpiakaalp
capitata
47 cathelicidin antimicrobial Homo sapiens
mktqrnghslgrwslvIllIglvmplaiiaqvlsykeavl
peptide raidginqrssdanlyrildldprptmdgdpdtpkpvsft
vkencprttqqspedcdfIckdgIv1,7cmgtvtlnqargs
fdiscdkdnkrfallgdffrkskekigkefIcrivqrikdf
CA 02 7 5 8 17 8 2 0 1 1-1 1-07
WO 2005/012492 PCT/US2004/024627
Table 1
Antimicrobial Peptides
SEQ ID Name Organism Sequence
NO:
linlvprtes
48 myeloid cathelicidin 3 Equus caballus
metqmtrclgrwsp111llgIvippattqalsykeavir
avdglnqrssclenlyrlleldplpkgdkdsdtpkpvsfmv
ketvcprimkqtpeqcdficenglykqcvgtvildpvkdyf
dascdepqrvIcrfhsvgsliqrhqqmirdkseatrhgni
itrpkIllas
49 myeloid antimicrobial Bos taurus
metqrasIsIgrwslwIllIglalpsasaqalsyreavIr
peptide BMAP-28
avdqlnelcsseanlyrlleldpppkeddenpnipkpvsfr
vketvcprtsqqspeqcdfkenglIkecvgtvtldqvgsn
fditcavpqsvgglislgrkilrawkkygpiivpiirig
50 myeloid cathelicidin 1 Equus cabalius
metqmtrclgrwsp11111glvippattqalsykeavh
avdglriqrssdenlyrIleldpIpkgdkdsdtpkpvsfmv
ketvcprirnkqtpeqedfIcenglvkqcygtvilgpvkdhf
dvscgepqrvlafgrlaksfinnrillprrkillas
51 SMAP 29 Ovis cries metqraslsIgreslwIlllglalpsasaqvlsyreavlr
aadqlneksseanlyrlleldpppkqddensnipkpvsfr
vketvcprtsqqpaeqcdflcengllkecvgtvfidqvrim
fditcaepqsvrglrrlgrkiahgvickygptvlriiriag
52 BNP-1 Bos taurus rIcrivvirvcr
53 HNP-1 HOMO sapiens acycripaciagerrygtciyqgrlwafcc
54 HNP-2 H01110 sapiens cycripaciagerrygtciyqgrlwafcc
55 HNP-3 H07110 sapiens dcycripaciagerrygtciyqgrlwafcc
56 HNP-4 Homo sapiens vcscrlyfcn-telrygncliggvsftycctrv
57 NP-1 07yetolagus vvcacrralclprerragfcrirgrihplccrr
c/a/jai/us
58 NP-2 Olyetolagus yvcacrralcIplerragfcrirgrihplccrr
cunicuhis
59 NP-3A 07ctolagus gicacurfepnserfsgyervngaryvrccsrr
cunicu/us
60 NP-3B Oryctolagus grevcrkqllcsyrerrigdckirgvrfpfccpr
cuniculus
61 NP-4 Olyctolagus vsctcrrfsegfgerasgsctvnggyrhticcrr
31
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Table 1
Antimicrobial Peptides
SEQ ID Name Organism Sequence
NO:
cuniculus
62 NP-5 Olyctolagus vfctcrgflegsgerasgsctingvrhticcrr
cuniculus
63 RatNP-1 Rattus vtcycntregfrer1sgacgyrgriyriccr
norvegicus
64 Rat-NP-3 Rattus cscrysscrfgerllsgacrIngriyrlcc
norvegicus
65 Rat-NP-4 Rattus actcrigacvsgerltgacgIngriyrIccr
norvegicus
66 GPNP Guinea pig rrcichrterfpyrrIgtcifqnrvytfcc
67 theta defensin-I Macaca rcictrgfcrcicrrgvc
nzuiatta
68 defensin CUAl Helianthus
mkssmlanfaalllvvarllanemggplvveartcesqslik
animus fkgtclsdtocanychserfsggkergfracfchhc
69 defensin SD2 Helianthus
mkssmkrnfaalllvvmcllanemggplvveartcesqshk
011111111S flgtelsdtncanychserfsggkcrgfirrcfctthc
70 neutrophil defensin 2 Macaca acycripaclagerrygtcfyingrywafcc
?nulatta
71 4 KDA defensin Androctortus
gfgcpfnqgachrhcrsiarggycaglfkqtetcyr
austratis hector
72 defensin Alyithis gfgcpnnyqchrhcksipgrcggycggxhrIrctcyrc
galloprovincialis
73 defensin AMP1 Heuchera duldcdvpsgtwsghcgssskcsqqckdrehfayggach
sanguinea yqfpsvkcfcicrqc
74 defensin AMP1 Clitoria nicerasltwtgnegntghcdtqcnnvesakhgachkrgn
ternatea wkcfcyfnc
75 cysteine-rich cryptdin-1 Mus muscri/us
mkklvIlfalvllafqvqadsiqntdeetkteeqpgekdq
32
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Table 1
Antimicrobial Peptides
SEQ ID Name Organism Sequence
NO:
homolog avsvsfgdpqgsalqdaalgwgrrcpqcprcpscpscprc
prcprckcnpk
76 beta-defensin-9 Bos taurus
qgvmfvtcrinrgfcvpircpglurqigtclgpqikccr
77 beta-defensin-7 Bos taurus
qgvmfvtcrinrgfcvpircpghrrqigtclgprikccr
'78 beta-defensin-6 Bos taunts
qgvmhvtcriyggfcvpircpgrtrqigtcfgrpvkccrrw
79 beta-defensin-5 Bos taunts
qvvrripqscrwrungvcipiscpgumrqigtcfgprvpccr
80 beta-defensin-4 Bos taurus
qrympqscrwrimgvcipflcrvgmrqigtcfgprvpccrr
81 beta-defensin-3 Bos taurus
qgvmhvtcrinrgfcvpircpgrtrqigtcfgprikccrsw
82 beta-defensin-10 Bos taunts
qgvrsylscwgnrgicllnrcpgrrnrqigtclaprvkccr
83 beta-defensin-13 Bos taurus
sgisgplscgrnggvcipircpvpnuqigtcfgrpvkccrsw
84 beta-defensin-1 Bos taurus
dfaschtnggiclpnrcpgluniqigicfrprvkccrsw
85 coleoptericin Zophobas slqggapnfpqpsqqnggwqvspdlgrddkgntrgqieiq
atratus nkgkdhdfnagwgIcvirgplakakptwhvggtyrr
86 defensin C Aedes aegypti
atedllsgfgvgdsacaahciargnrggycnskkvcvern
87 defensin B edulis gfgcpndypchrhcksipgryggycggxhr1rctc
88 sapecin C Sarcophaga
atcdllsgigvqhsacalhcvfrgnrggyctgkgicvem
peregrina
89 macrophage antibiotic Otyctolagus
mrtlallaaillvalqaqaehvsysidevvdqqppqaedq
peptide MCP-1 cunicuhts
dvaiyvkehessalealgvkagyveacrralelprerrag
fairgriliplcca
90 cryptdin-2 Mus inuscuhts
mkplyllsalvlIsfqvqadpiqntdeetkteeqsgeedq
avsvsfgdregaslqeeslrdIvcycrtrgclurernmgt
crkghlrnyticc
91 cryptdin-5 Mus inuscidus
mIctfyllsalvllafqvqadpihktdeetnteeqpgeedq
avsisfggqegsalheelskklicycrirgclarervfgt
cmlfltfvfccs
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Table 1
Antimicrobial Peptides
SEQ ID Name Organism Sequence
NO:
92 cryptdin 12 MUS 112USCUILIS
1rdlycycrargckgrermngterkghllyrnlccr
93 defensin Pyrrhocoris
atcdilsfqsqwvtpnhagcallicvikgykggqckitychcrr
apterus
94 defensin R-5 Rattus vtcycrstrcgfrerlsgacgyrgriyrlccr
notTegicus
95 defensin R-2 Rattus vtcscrtsscrfgerlsgactingdyrlcc
norvegicra
96 defensin NP-6 Otyctolagus gicacurfelnfeqfsgycnragaryvrccsrr
cuniculus
97 beta-defensin-2 Pan troglodytes
mrvlyllfsflfiflmplpgvfggisdpvtclksgaichp
vfcprrykqigtcglpgtkcckkp
98 beta-defensin-1 Capra hircus
mahhIllvlfflvlsagsgftqgirsrrschmkgveal
trcpmrnrqigtcfgppvkccrkk
99 beta defensin-2 Capra hircus
mrlhhlllalfrIvlsagsgftqgiinluscyrnkgvcap
arcpmmrqigtchgppvkccrkk
100 defensin-3 Macaca mrtivilaaillvalqaqaeplqartdeataaqeqiptdn
mulatta pevvvslawdeslapkdsvpglrhunacycripaclager
rygtcfyrrrywafcc
101 defensin-1 Macaca mrtivilaaillvalqaqaeplqartdeataaqeqiptdn
mulatta
pevvvslawdeslapkdsvpgIrkrunacycripaclager
rygtcfylgrvwafcc
102 neutrophi1 defensin 1 Mesocricettis
vtcfcragcasrerhigycrfgntiyrIccrr
aziratus
103 neutrophil defensin 1 Mesocricefus
cfcicmvcdsgetqigycrlgntfyrlccrq
auratus
104 Gallinacin 1-alpha Gallus gallus
grksdcfrkngfcafIkcpyltlisgkcsrfkleckriw
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Table 1
Antimicrobial Peptides
SEQ ID Name Organism Sequence
NO:
105 defensin Allomyrina
vtccIllsfeakgfaanlislcaahclaigrrggscergvcicrr
dichotoma
106 neutrophil cationic Cavia porcellus
acicttricrfpyrrlgtcifqnrvytfcc
peptide 1
Accordingly, in some embodiments the present invention contemplates the
production of keratinocytes and skin equivalents expressing an antimicrobial
polypeptide,
and compositions and methods for making keratinocytes expressing an exogenous
antimicrobial polypeptide. In preferred embodiments, the antimicrobial
polypeptide is a
defensin or a cathelicidin. In still more preferred embodiments, the defensin
is a human
beta defensin. In still more preferred embodiments, the human beta defensin is
human beta
defensin 1, 2 or 3. In some embodiments, the keratinocytes are transfected
with more than
one defensin selected from the group consisting of human beta-defensin 1, 2 or
3. In
preferred embodiments, keratinocytes are induced to express an
antimicrobialpolypeptide
through transfection with an expression vector comprising a gene encoding an
antimicrobial
polypeptide. An expression vector coinprising a gene encoding an antimicrobial
polypeptide can be produced by operably linking an antimicrobial polypeptide
coding
sequence to one or more regulatory sequences such that the resulting vector is
operable in a
desired host.
In preferred embodiments, the antimicrobial polypeptide is isolated from a DNA
source, cloned, sequenced, and incoiporated into a selection vector. In
certain
embodiments, isolation of the antimicrobial polypeptide DNA occurs via PCR by
using
primer sequences designed to amplify the antimicrobial polypeptide sequence.
Primer
sequences specific for the desired antimicrobial polypeptide may be obtained
from
Genbank. Amplification of a DNA source with such primer sequences through
standard
PCR procedures results in antimicrobial polypeptide cDNA isolation. In
preferred
embodiments, the source of cDNA is human cDNA.
CA 02758178 2011-11-07
WO 2005/012492 PCT/US2004/024627
D) Methods of Generating host cells expressing exogenous polypeptides
In some embodiments, the present invention provides methods of generating host
cells (e.g., keratinocytes) and skin equivalents expressing one or more
exogenous
polypeptides (e.g., KGF-2 and/or antimicrobial polypeptides. The present
invention is not
limited to particular methods for the generation of such cells and skin
equivalents.
Exemplary methods are described below. Additional methods are known to those
skilled in
the relevant arts.
In certain embodiments, the antimicrobial polypeptide cDNA is cloned into a
cloning vector. A regulatory sequence that can be linked to the antimicrobial
polypeptide
DNA sequence in an expression vector is a promoter that is operable in the
host cell in
which the antimicrobial polypeptide is to be expressed. Optionally, other
regulatory
sequences can be used herein, such as one or more of an enhancer sequence, an
intron with
functional splice donor and acceptance sites, a signal sequence for directing
secretion of the
defensin, a polyadenylation sequence, other transcription terminator
sequences, and a
sequence homologous to the host cell genome. Other sequences, such as origin
of
replication, can be added to the vector as well to optimize expression of the
desired
defensin. Further, a selectable marker can be present in the expression vector
for selection
of the presence thereof in the transformed host cells.
In preferred embodiments, antimicrobial polypeptide is fused to a regulatory
sequence that drives the expression of the polypeptide (e.g., a promoter). In
preferred
embodiments, the regulatory sequence is the involucrin promoter (SEQ NO: 12)
or the
keratin-14 promoter. However, any promoter that would allow expression of the
antimicrobial polypeptide in a desired host can be used in the present
invention.
Mammalian promoter sequences that can be used herein are those from manunalian
viruses
that are highly expressed and that have a broad host range. Examples include
the SV40
early promoter, the Cytomegalovirus ("CMV") immediate early promoter mouse man-
unary
tumor virus long terminal repeat ("LTR") promoter, adenovirus major late
promoter (Ad
MLP), and Herpes Simplex Virus ("HSV") promoter. In addition, promoter
sequences
derived from non-viral genes, such as the murine metallothionein gene,
ubiquitin and
elongation factor alpha (EF-1a) are also useful herein. These promoters can
further be either
constitutive or regulated, such as those that can be induced with
glucocorticoids in
hormone-responsive cells.
In some preferred embodiments, host cells (e.g., keratinocytes cells)
expressing
KGF-2 or antimicrobial polypeptides can be produced by conventional gene
expression
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WO 2005/012-192 PCT/US2004/024627
technology, as discussed in more detail below. The practice of the present
invention will
employ, unless otherwise indicated, conventional techniques of molecular
biology,
microbiology, recombinant DNA, and immunology, which are within the skill of
the art.
Such techniques are explained fully in the literature, including Sambrook, et
al.,
MOLECULAR CLONING: A LABORATORY MANUAL 2nd ed. (Cold Spring Harbor
Laboratory Press, 1989); DNA CLONING, Vol. I and 11, D. N Glover ed. (IRL
Press,
1985); OLIGONUCLEOTIDE SYNTHESIS, M. J. Gait ed. (IRL Press, 1984); NUCLEIC
ACID HYBRIDIZATION, B. D. Hames & S. J. Higgins eds. (IRL Press, 1984);
TRANSCRIPTION AND TRANSLATION, B. D. Hames & S. J. Higgins eds., (IRL Press,
1984); ANIMAL CELL CULTURE, R. I. Freshney ed. (IRL Press, 1986); IMMOBILIZED
CELLS AND ENZYMES, K. Mosbach (IRL Press, 1986); B. Perbal, A PRACTICAL
GUIDE TO MOLECULAR CLONING, Wiley (1984); the series, METHODS IN
ENZYMOLOGY, Academic Press, Inc.; GENE TRANSFER VECTORS FOR
MAMMALIAN CELLS, J. H. Miller and M. P. Calos eds. (Cold Spring Harbor
Laboratory,
1987); METHODS IN ENZYMOLOGY, Vol. 154 and 155, Wu and Grossman, eds., and
Wu, ed., respectively (Academic Press, 1987), IMMUNOCHEMICAL METHODS IN
CELL AND MOLECULAR BIOLOGY, R. J. Mayer and J. H. Walker, eds. (Academic
Press London, Harcourt Brace U.S., 1987), PRO FEIN PURIFICATION: PRINCIPLES
AND PRACTICE, 2nd ed. (Springer-Verlag, N.Y. (1987), and HANDBOOK OF
EXTERIMENTAL IMMUNOLOGY, Vol. I-IV, D. M. Weir et al., (Blackwell Scientific
Publications, 1986); Kitts et al., Biotechniques 14:810-817 (1993); Munemitsu
et al., Mol.
and Cell. Biol. 10:5977-5982 (1990).
The present invention contemplates keratinocytes and skin equivalents
expressing
KGF-2 and/or antimicrobial polypeptides, and compositions and methods for
making such
cells. In some embodiments, host cells are induced to express exogenous
polypeptides
through transfection with an expression vector containing DNA encoding the
exogenous
polypeptide. An expression vector containing KGF-2 DNA can be produced by
operably
linking KGF-2 to one or more regulatory sequences such that the resulting
vector is
operable in a desired host. Cell transformation procedures suitable for use
herein are those
known in the art and include, for example with mammalian cell systems, dextran-
mediated
transfection, calcium phosphate precipitation, polybrene-mediated
transfection, protoplast
fusion, electroporation, encapsulation of the exogenous polynucleotide in
liposomes, and
direct microinjection of the DNA into nuclei. In preferred embodiments, cells
are
37
CA 02758178 2011-11-07
transfected with a pUB-Bsd expression vector containing exogenous DNA (e.g.,
KGF-2 and
antimicrobial polypeptides) operably linked to promoter (e.g., K14 or
involucrin) DNA.
Immunoassays and activity assays that are known in the art can be utilized
herein to
deterrnine if the transformed host cells are expressing the desired exogenous
polypeptide
(e.g., KGF-2 and antimicrobial polypeptides). In some embodiments, detection
of
intracellular production of KGF-2 or antimicrobial polypeptides by transformed
host cells is
accomplished with an immunofluorescence assay. In preferred embodiments,
detection of
intracellular production of exogenous polypeptides by transformed host cells
is
accomplished through a RT-PCR screen. In further embodiments, detection of
secreted or
extracellular production of KGF-2 or antimicrobial polypeptides by transformed
host cells is
accomplished through a direct ELISA screen. In some embodiments, the KGF-2 or
antimicrobial polypeptide is detected by Western blotting.
In other embodiments, expression vectors comprising exogenous polypeptides are
introduced directly into tissues (e.g., human skin equivalents). Expression
vectors may be
introduced into tissues using any suitable technique including, but not
limited to,
electroporation, particle bombardment (e.g., 6,685,669, 6,592,545, and
6,004,286) and transfection.
11. Selection of cells by Electroporation
Experiments conducted during the course of development of the present
invention
(See e.g., Example 26) resulted in the identification of a novel technique for
the selection of
cells within a population. The experiments demonstrated that cells
electroporated in the
presence or absence of exogenous nucleic acid and selection demonstrated
properties of
multipotency. Accordingly, in some embodiments, the present invention provides
methods
of selecting for cells in a population having desired growth and proliferation
properties.
In some embodiments, electroporation is used to select for cells with enhanced
pluripotency or multipotency. In other embodiments, electroporation is used to
select for
cells with enhanced pluripotency or multipotency. As used herein, the term
"pluripotent"
means the ability of a cell to differentiate into the three main genii layers:
endoderm,
ectoderm, and mesodertn. In some einbodiments, the cells with enhanced
pluripotency or
multipotency exhibit stem cells like properties.
For example, in some embodiments,' electroporation is used to select for cells
with
stein-cell like properties. Stem cells are undifferentiated cells that can
give rise to a
38
CA 02758178 2011-11-07
succession of mature functional cells. Stem cells can by embryonically derived
(See e.g.,
U.S. Pat. Nos. 5,843,780 and 6,200,806) or derived from adult cells.
Examples of adult stem cells include hematopoietic stem cells,
neural stem cells, mesenchymal stem cells, and bone marrow stromal cells.
These stein
cells have demonstrated the ability to differentiate into a variety of cell
types including
adipocytes, chondrocytes, osteocytes, myocytes, bone marrow stromal cells, and
thymic
stroma (mesenchymal stem cells); hepatocytes, vascular cells, and muscle cells
(hematopoietic stern cells); myocytes, hepatocytes, and glial cells (bone
marrow stoma]
cells) and, cells from all three germ layers (adult neural stern cells).
In other embodiments, electroporation is used to select for cells with
extended
proliferative capacity. For example, experiments conducted during the course
of
development of the present invention demonstrated that electroporated cells
were typically
the larger surviving colonies.
In yet other embodiments, electroporation is used to select for keratinocytes
having
holoclone or meroclone cell morphology (e.g., a colony morphology of tightly
packed,
uniform cells, smooth colony edges, overall round colony morphology).
111. Treatment of wounds with keratinocytes cells transfected with exogenous
polypeptides
Successful treatment of chronic skin wounds (e.g., venous ulcers, diabetic
ulcers,
pressure ulcers) is a serious problem. The healing of such a wound often times
takes well
over a year of treatment. Treatment options currently include dressings and
debridement
(use of chemicals or surgery to clear away necrotic tissue), and/or
antibiotics in the case of
infection. These treatment options take extended periods of time and high
amounts of
patient compliance. As such, a therapy that can increase a practioner's
success in healing
chronic wounds and accelerate the rate of wound healing would meet an unmet
need in the
In some embodiments, the present invention contemplates treatment of skin
wound
with keratinocytes and skin equivalents expression exogenous antimicriobial
and/or KGF-2
polypeptides.
KGF-2 is associated with skin wound healing. In skin, KGF-2 is naturally
expressed in the dermal compartment. Topical application of KGF-2 to skin
wounds
increases dermal cell proliferation. In addition, KU-2 manifests strong
mitogenic activity
39
CA 02758178 2011-11-07
in dermal cells and stimulates granulation tissue foimation in full thickness
excisional
wounds. KGF-2 accelerated wound closure is transient and does not cause scar
formation
after complete wound healing (Yu-Ping et al. 1999). Local protein
administration, however,
has been shown to be ineffective due to enzymes and proteases in the wound
fluid (jeschke
et al. 2002). KGF-2 selectively induces normal epithelial cell proliferation,
differentiation
and migration, while having no in vitro or in vivo proliferative effects on
KGFR (+) human
epithelial-like tumors. (Alderson et al. 2002). As such, KGF-2 is an
attractive candidate for
therapeutic use to enhance wound healing.
The present invention contemplates treatment of skin wounds with keratinocytes
or
skin equivalents expressing KGF-2 and/or antimicrobial polypeptides. In some
embodiments, cells expressing KGF-2 and/or antimicrobial polypeptides are
topically
applied to wound sites. In some embodhnents, the keratinocytes are applied via
a spray,
while in other embodiments, the keratinocytes are applied via a gel. In other
embodiments,
cells expressing KGF-2 and/or antimicrobial polypeptides are used for
eng,raftment on
partial thickness wounds. In other embodiments, cells expressing KGF-2 and/or
antimicrobial polypeptides are used for engraftment on full thickness wounds.
In other
embodiments, cells expressing KGF-2 and/or antimicrobial polypeptides are used
to treat
numerous types of internal wounds, including, but not limited to, internal
wounds of the
mucous membranes that line the gastrointestinal tract, ulcerative colitis, and
inflammation
of mucous membranes that may be caused by cancer therapies. In still other
embodiments,
cells expressing KGF-2 and/or antimicrobial polypeptides are used as a
temporary or
permanent wound dressing.
Cells expressing KGF-2 and/or antimicrobial polypeptides find use in wound
closure
and burn treatment applications. The use of autografts and allografts for the
treatment of
bums and wound closure is described in Myers et al., A. J. Surg. 170(1):75-83
(1995) and
U.S. Pat. Nos. 5,693,332; 5,658,331; and 6,039,760.
In some embodiments, the skin equivalents may be used in conjunction with
dermal replacements such as DERMAGRAFT. In other embodiments, the skin
equivalents
are produced using both a standard source of keratinocytes (e.g., NIKS cells)
and
keratinocytes from the patient that will receive the graft. Therefore, the
skin equivalent
contains keratinocytes from two different sources, in still further
embodiments, the skin
equivalent contains keratinocytes from a human tissue isolate. Accordingly,
the present
invention provides methods for wound closure, including wourids caused by
bums,
comprising providing cells expressing KGF-2 and/or antimicrobial polypeptides
and a
CA 02758178 2011-11-07
patient suffering from a wound and treating the patient with the cells under
conditions such
that the wound is closed.
Detailed methods for producing the skin equivalents of the present invention
are
disclosed in the following Experimental section. However, the present
invention is not
limited to the production of skin equivalents by the methods. Indeed, a
variety of
organotypic culture techniques may be used to produce skin equivalents,
including those
described in U.S. Pat. Nos. 5,536,656 and 4,485,096.
In some embodiments, different populations of keratinocytes are used to
construct the skin equivalent. Accordingly, in some embodiments, the skin
equivalents of
the present invention are formed from keratinocytes derived from an
immortalized cell line
(e.g., NIKS cells) and cell derived from a patient. In other embodiments, the
skin
equivalents of the present invention are formed from at least a first
population of
keratinocytes derived from an inunortalized cell line that express a exogenous
antimicrobial
polypeptide and/or KGF-2 and a second population of keratinocytes derived from
an
immortalized cell line that do not express a exogenous antimicrobial
polypeptide. It is
contemplated that varying the ratio of the two populations the dose of
antimicrobial
polypeptide and/or KGF-2 delivered can be varied. In still other embodiments,
the skin
equivalents are foinied from at least a first population of keratinocytes
expressing a first
exogenous antimicrobial polypeptide (e.g., hBD-1) and at least a second
population of
keratinocytes expressing a second exogenous antimicrobial polypeptide (e.g.,
1113D-2 or
hBD-3). Again, the ratios of the cell populations can be varied to vary the
dose. In still
other embodiments, the skin equivalents are formed from at least a first
population of
keratinocytes expressing a first exogenous antimicrobial polypeptide (e.g.,
hBD-1), at least
a second population of keratinocytes expressing a second exogenous
antimicrobial
polypeptide (e.g., 11BD-2 or hBD-3), and keratinocytes derived fiona a
patient.
In a further embodiment, the KGF-2 and/or antimicrobial polypeptide or a
conjugate
thereof can be mixed with a pharmaceutically acceptable carrier to produce a
therapeutic
composition that can be administered for therapeutic purposes, for example,
for wound
healing, and for treatment of hyperproliferative diseases of the skin and
tumors, such as
psoriasis and basal cell carcinoma.
Iii still further embodiments, the cells expressing KGF-2 and/or antimicrobial
polypeptides are engineered to provide a therapeutic agent to a subject. The
present
invention is not limited to the delivery of any particular therapeutic agent.
Indeed, it is
contemplated that a variety of therapeutic agents may be delivered to the
subject, including,
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WO 20051012492 PCT/US2004/024627
but not limited to, enzymes, peptides, peptide hormones, other proteins,
ribosomal RNA,
ribozymes, and antisense RNA. These therapeutic agents may be delivered for a
variety of
purposes, including but not limited to the purpose of correcting genetic
defects. In some
particular preferred embodiments, the therapeutic agent is delivered for the
purpose of
detoxifying a patient with an inherited inborn error of metabolism (e.g.,
aninoacidopathesis)
in which the graft serves as wild-type tissue. It is contemplated that
delivery of the
therapeutic agent corrects the defect. In some embodiments, the cells
expressing KGF-2
and/or antimicrobial polypeptides are transfected with a DNA construct
encoding a
therapeutic agent (e.g., insulin, clotting factor IX, erythropoietin, etc) and
the cells grafted
onto the subject. The therapeutic agent is then delivered to the patient's
bloodstream or
other tissues from the graft. In preferred embodiments, the nucleic acid
encoding the
therapeutic agent is operably linked to a suitable promoter. The present
invention is not
limited to the use of any particular promoter. Indeed, the use of a variety of
promoters is
contemplated, including, but not limited to, inducible, constitutive, tissue
specific, and
keratinocyte specific promoters. In some embodiments, the nucleic acid
encoding the
therapeutic agent is introduced directly into the keratinocytes (i.e., by
calcium phosphate co-
precipitation or via liposome transfection). In other preferred embodiments,
the nucleic
acid encoding the therapeutic agent is provided as a vector and the vector is
introduced into
the keratinocytes by methods known in the art. In some embodiments, the vector
is an
episomal vector such as a plasmid. In other embodiments, the vector integrates
into the
genome of the keratinocytes. Examples of integrating vectors include, but are
not limited
to, retroviral vectors, adeno-associated virus vectors, and transposon
vectors.
IV. Testing Methods
The host cells and cultured skin tissue of the present invention may be used
for a
variety of in vitro tests. In particular, the host cells and cultured skin
tissue find use in the
evaluation of: skin care products, drug metabolism, cellular responses to test
compounds,
wound healing, phototoxicity, dermal irritation, dermal inflammation, skin
corrosivity, and
cell damage. The host cells and cultured skin tissue are provided in a variety
of fonnats for
testing, including 6-well, 24-well, and 96-well plates. Additionally, the
cultured skin tissue
can be divided by standard dissection techniques and then tested. The cultured
skin tissue
of the present invention may have both an epidermal layer with a
differentiated stratum
corneum and dennal layer that includes dermal fibroblasts. As described above,
in
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WO 2005/012492 PCT/US2004/024627
preferred embodiments, the epidermal layer is derived from immortalized NIKS
cells.
Other preferred cell lines, including NIKS cells are characterized by; i)
being immortalized;
ii) being nontumorigenic; iii) forming cornified envelopes when induced to
differentiate; iv)
undergoing normal squamous differentiation in organotypic culture; and v)
maintaining cell
type-specific growth requirements, wherein said cell type-specific growth
requirements
include 1) exhibition of morphological characteristics of nonnal human
keratinocytes when
cultured in standard keratinocyte growth medium in the presence of mitomycin C-
treated
3T3 feeder cells; 2) dependence on epidermal growth factor for growth; and 3)
inhibition of
growth by transforming growth factor 1.
The present invention encompasses a variety of screening assays. In some
embodiments, the screening method comprises providing a host cell or cultured
skin tissue
of the present invention and at least one test compound or product (e.g., a
skin care product
such as a moisturizer, cosmetic, dye, or fragrance; the products can be in any
from,
including, but not limited to, creams, lotions, liquids and sprays), applying
the product or
test compound to the host cell or cultured skin tissue, and assaying the
effect of the product
or test compound on the host cell or cultured skin tissue. A wide variety of
assays are used
to determine the effect of the product or test compound on the cultured skin
tissue. These
assays include, but are not limited to, MTT cytotoxicity assays (Gay, The
Living Skin
Equivalent as an In Vitro Model for Ranking the Toxic Potential of Dermal
Irritants, Toxic.
In Vitro (1992)) and ELISA to assay the release of inflammatory modulators
(e.g.,
prostaglandin E2, prostacyclin, and interleukin-l-alpha) and chemoattractants.
The assays
can be further directed to the toxicity, potency, or efficacy of the compound
or product.
Additionally, the effect of the compound or product on growth, barrier
function, or tissue
strength can be tested.
In particular, the present invention contemplates the use of host cells or
cultured skin
tissue for high throughput screening of compounds from combinatorial libraries
(e.g.,
libraries containing greater than 104 compounds). In some embodiments, the
cells are used
in second messenger assays that monitor signal transduction following
activation of cell-
surface receptors. In other embodiments, the cells can be used in reporter
gene assays that
monitor cellular responses at the transcription/translation level. In still
further
embodiments, the cells can be used in cell proliferation assays to monitor the
overall
growth/no growth response of cells to external stimuli.
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In second messenger assays, host cells or cultured skin tissue is treated with
a
compound or plurality of compounds (e.g., from a combinatorial library) and
assayed for
the presence or absence of a second messenger response. In some preferred
embodiments,
the cells (e.g., NIKS cells) used to create cultured skin tissue are
transfected with an
expression vector encoding a recombinant cell surface receptor, ion-channel,
voltage gated
channel or some other protein of interest involved in a signaling cascade. It
is contemplated
that at least some of the compounds in the combinatorial library can serve as
agonists,
antagonists, activators, or inhibitors of the protein or proteins encoded by
the vectors. It is
also contemplated that at least some of the compounds in the combinatorial
library can
serve as agonists, antagonists, activators, or inhibitors of protein acting
upstream or
downstream of the protein encoded by the vector in a signal transduction
pathway.
In some embodiments, the second messenger assays measure fluorescent signals
from reporter molecules that respond to intracellular changes (e.g., Ca2+
concentration,
membrane potential, pH, 1P3, cAMP, arachidonic acid release) due to
stimulation of
membrane receptors and ion channels (e.g., ligand gated ion channels; see
Denyer et
al., Drug Discov. Today 3:323-32 [19981; and Gonzales et al., Drug. Discov.
Today
4:431-39 [1999]). Examples of reporter molecules include, but are not limited
to,
FRET (florescence resonance energy transfer) systems (e.g., Cuo-lipids and
oxonols,
EDAN/DABCYL), calcium sensitive indicators (e.g., Fluo-3, FURA 2, IN1D0 1, and
FLU03/AM, BAPTA AM), chloride-sensitive indicators (e.g., SPQ, SPA), potassium-
sensitive indicators (e.g., PBFI), sodium-sensitive indicators (e.g., SBFI),
and pH
sensitive indicators (e.g., BCECF).
In general, the cells comprising cultured skin tissue are loaded with the
indicator
prior to exposure to the compound. Responses of the host cells to treatment
with the
compounds can be detected by methods known in the art, including, but not
limited to,
fluorescence microscopy, confocal microscopy (e.g., FCS systems), flow
cytometry,
microfluidic devices, FLIPR systems (See, e.g., Schroeder and Neagle, J.
Biomol. Screening
1:75-80 [1996]), and plate-reading systems. In some preferred embodiments, the
response
(e.g., increase in fluorescent intensity) caused by compound of unknown
activity is
compared to the response generated by a known agonist and expressed as a
percentage of
the maximal response of the known agonist. The maximum response caused by a
known
agonist is defined as a 100% response. Likewise, the maximal response recorded
after
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addition of an agonist to a sample containing a known or test antagonist is
detectably lower
than the 100% response.
The host cells and cultured skin tissue of the present invention are also
useful in
reporter gene assays. Reporter gene assays involve the use of host cells
transfected with
vectors encoding a nucleic acid comprising transcriptional control elements of
a target gene
(i.e., a gene that controls the biological expression and function of a
disease target or
inflammatory response) spliced to a coding sequence for a reporter gene.
Therefore,
activation of the target gene results in activation of the reporter gene
product. This serves as
indicator of response such an inflammatory response. Therefore, in some
embodiments, the
reporter gene construct comprises the 5' regulatory region (e.g., promoters
and/or
enhancers) of a protein that is induced due to skin inflammation or irritation
or protein that
is involved in the synthesis of compounds produced in response to inflammation
or
irritation (e.g., prostaglandin or prostacyclin) operably linked to a reporter
gene. Examples
of reporter genes finding use in the present invention include, but are not
limited to,
chloramphenicol transferase, alkaline phosphatase, firefly and bacterial
luciferases, p-
galactosidase, p-lactamase, and green fluorescent protein. The production of
these
proteins, with the exception of green fluorescent protein, is detected through
the use of
chemiluminescent, colorimetric, or bioluminecent products of specific
substrates (e.g., X-
gal and luciferin). Comparisons between compounds of known and unknown
activities may
be conducted as described above.
In other preferred embodiments, the host cells or cultured skin tissue find
use for
screening the efficacy of drug introduction across the skin or the affect of
drugs directed to
the skin. In these embodiments, cultured skin tissue or host cells are treated
with the drug
delivery system or drug, and the permeation, penetration, or retention or the
drug into the
skin equivalent is assayed. Methods for assaying drug permeation are provided
in Asbill et
al., Pharm Res. 17(9): 1092-97 (2000). In some embodiments, cultured skin
tissue is
mounted on top of modified Franz diffusion cells. The cultured skin tissue is
allowed to
hydrate for one hour and then pretreated for one hour with propylene glycol. A
saturated
suspension of the model drug in propylene glycol is then added to the cultured
skin tissue.
The cultured skin tissue can then be sampled at predeterrnined intervals. The
cultured skin
tissue is then analyzed by HPLC to determine the concentration of the drug in
the sample.
Log P values for the drugs can be determined using the ACD program (Advanced
CA 02758178 2011-11-07
Chemistry Inc., Ontario, Canada). These methods may be adapted to study the
delivery of
drugs via transdermal patches or other delivery modes.
lt is contemplated that cultured skin tissue of the present invention is also
useful for
the culture and study of tumors that occur naturally in the skin as well as
for the culture and
study of pathogens that affect the skin. Accordingly, in some embodiments, it
contemplated
that the cultured skin tissue of the present invention is seeded with
malignant cells. By way
of non-limiting example, the cultured skin tissue can be seeded with malignant
SCC13y
eells as described in U.S. Pat. No. 5,989,837 to
provide a model of human squamous cell carcinoma. These seeded cultured skin
tissue can
then be used to screen compounds or other treatment strategies (e.g.,
radiation or
tornotherapy) for efficacy against the tumor in its natural environment. Thus,
some
enibodinients of the present invention provide methods comprising providing
cultured skin
tissue comprising malignant cells or a tumor and at least one test compound,
treating the
cultured skin tissue with the compound, and assaying the effect of the
treatment on the
malignant cells or tumors. In other embodiments of the present invention,
methods are
provided that comprise providing cultured skin tissue comprising malignant
cells or a tumor
and at least one test therapy (e.g., radiation or phototherapy, treating the
cultured skin tissue
with the therapy, and assaying the effect of the therapy on the malignant
cells or tumors.
In other embodiments, cultured skin tissue is used to culture and study skin
pathogens. By way of non-limiting example, cultured skin tissue is infected
with human
papilloma virus (HPV) such as HPV18. Methods for preparing cultured skin
tissue infected
with HPV are described in U.S, Pat. No. 5,994,115.
Thus, some embodiments of the present invention provide methods comprising
providing cultured skin tissue infected with a pathogen of interest and at
least one test
conapoimd or treatment and treating the cultured skin tissue with the test
compound or
treatment. In some preferred embodiments, the methods further comprise
assaying the
effect the test coinpound or treatment on the pathogen. Such assays may he
conducted by
assaying the presence, absence, or quantity of the pathogen in the cultured
skin tissue
following treatment. For example, an ELISA may be performed to detect or
quantify the
pathogen. In some particularly prefeired embodiments, the pathogen is viral
pathogen such
as HPV.
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EXPERIMENTAL
The following examples are provided in order to demonstrate and further
illustrate
certain preferred embodiments and aspects of the present invention and are not
to be
construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations
apply: eq
(equivalents); M (Molar); u.M (micromolar); N (Normal); mol (moles); mmol
(millimoles);
gmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); lig
(micrograms); ng
(nanograms); 1 or L (liters); ml (milliliters); ul (microliters); cm
(centimeters); mm
(millimeters); p.m (micrometers); nm (nanometers); C (degrees Centigrade); U
(units), mU
(milliunits); min. (minutes); sec. (seconds); % (percent); kb (kilobase); bp
(base pair); PCR
(polymerase chain reaction); BSA (bovine serum albumin); Pfu (Pyrococcus
furiosus).
Example 1
Keratin 14 Promoter Cloning and Characterization
This Example describes the method used to isolate, clone and characterize the
K14
promoter DNA. Primer sequences were designed based on the published K14
Promoter
sequence available at Genbank (Genbank Accession #U11076). In order to amplify
the
2.35kb full length K14 promoter sequence, the following PCR primers were used:
Fwd 5'-AAGCTTATATTCCATGCTAGGGTTCTG-3' (ST080)(SEQ JD NO:1)
Rev 5'-GGTGCAGAGGAGGGAGGTGAGCGA-3' (ST081) (SEQ ID NO:2)
Human genomic DNA (Promega) was amplified with these primers using Amplitaq
DNA polymerase (Promega). Following a denaturation at 95 C for 4 minutes,
samples
were subjected to the following for 30 cycles: denaturation at 95 C for 1
minute, annealing
conditions at 58 C for 1 minute, extension at 72 C for 3 minutes. A final
extension at 72 C
for 7 minutes was followed by a 4 C hold. The expected PCR product of 2.35kb
was
observed. This PCR product was gel purified and subsequently used for cloning
into a TA
cloning vector. The pCR 2.1-TOPO TA Cloning Kit (Invitrogen/LifeTeelmologies)
was
used according to the standard protocol conditions.
Although thorough sequencing of this promoter has been problematic (typically
encountered when sequencing promoter regions presumably due to the high GC
content),
the cloned promoter sequence is different than the published K14 promoter
sequence
(Genbank sequence Accession #U110776). The consensus sequence of the cloned
Keratin
14 promoter fragment (SEQ ID NO:3) is provided in Figure la.
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In order to confirm the functionality of the K14 promoter sequence, a
luciferase
reporter gene expression system was used. The K14 promoter fragment was
shuttled into
the Hind III site of the pGL3 firefly luciferase vector multiple cloning site.
After
subcloning this full length K14 promoter Hind ICI fragment an opportunity to
truncate the
promoter fragment by approximately 300bp was easily accomplished using a
single Sma I
restriction enzyme site upstream in the multiple cloning site to release a
300bp 5' promoter
fragment. Published experiments demonstrate a similar 5' truncation of the K14
promoter
reduces the promoter activity by about 30% (Leask et al., Genes Dev. 4(10:1985-
1998
(1990)). The full length promoter fragment (2.3kb) firefly luciferase activity
was compared
to that of the 5' truncated Promoter fragment (-2.0kb) activity. The K14
Promoter
Luciferase Vector Construction is described in Figure 2
Results of luciferase reporter gene expression are as follows. The co-
expression of
Renilla Luciferase was used to correct for any variability introduced by
potentially different
transfection efficiencies or possible differences in cell numbers. After
normalization, the
firefly luciferase reporter gene results demonstrate strong promoter activity
from the full
length (2.3kb) K14 promoter fragment and approximately a 30% reduction in
firefly
luciferase activity in the truncated promoter fragnent This result is
consistent with that
reported by Leask et al.
Next, the full length K14 promoter was shuttled into the blasticidin selection
vector.
Example 2
KGF-2 Cloning and Characterization
This Example describes the isolation, cloning and characterization of KGF-2.
Primer sequences were designed based on the published KGF-2 sequence available
at
Genbank. In order to amplify the 627bp full length KGF-2 sequence, the
following PCR
primer sequences (BamH I-EcoR V) were used:
Fwd 5'-CGCGGATCCGCGATGTGGAAATGGATACTG-3' (ST127)(SEQ ID NO: 4)
Rev 5'-GGGATATCCTATGAGTGTACCACCATTGGA-3' (ST128)(SEQ ED NO:5)
Pfu Turbo DNA Polyinerase (Stratagene) was used to minimize the risk of PCR
induced errors. Human Universal QUICK-Clone cDNA (CLONTECH) was used as the
template for PCR amplification of the full length KGF-2 cDNA. Following a
denaturation
at 94 C for 4 minutes, samples were subjected to the following for 30 cycles:
denaturation
48
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at 94 C for 30 seconds, annealing conditions at 51 C for 30 seconds, extension
at 72 C for
1 minute. A final extension at 72 C for 7 minutes was followed by a 4 C Hold.
The
expected PCR product of 627bp was observed. After amplification, the addition
of 3' A-
overhangs to the Pfu PCR product was necessary to allow for efficient TA
cloning. The
PCR product was gel purified using a Matrix Gel Extraction Sys
teirirtivlarligen BioScience
Inc.). Gel purified PCR products were cloned into a commercially available TA
cloning kit.
TM
The pCR 2.1-TOPO TA Cloning Kit cinvitrogen/Lifetechnologies) was used
according to
the standard protocol conditions.
Sequencing reactions were performed using each of the two sequencing primers
that
1.0 span the cloning site (M13 forward and reverse primers). Additionally,
overlapping
sequence was obtained using the KGF-2 specific primers used to PCR amplify the
cDNA
(Primers ST127 and ST128). The cDNA sequence was identical to Genbank
accession
41167918.
Next, the TA cloned KGF-2 cDNA was shuttled into a pIR_ES vector. The TA clone
containing the correct KGF-2 cDNA sequence was digested with Banal-ll (5') and
EcoRV
(3') to release a 627bp KGF-2 cDNA. This product was cloned directly into the
BamIll and
EcoRV sites of the mammalian expression pIRESpuro clonal selection vector.
A re-amplification and TA cloning step was necessary to obtain the desired
restriction enzyme sites for directional cloning into tbe pUB-Bscl clonal
selection vector.
The primer sequences used to amplify the KGF-2 cDNA that contain the Not I and
Sal I
restriction enzyme sites are as follows.
Fwd 5'-GCGGCCGCATGTGGAAATGGATACTG-3' (ST133) (SEQ 111)
=
NO:109)
= Rev 5'-GTCGACCTATGAGTGTACCACCATTGGA-3' (ST134) (SBQ ID
NO:110)
The PCR conditions were the same as listed above. Pfu polymerase (Stratagene)
was used, but fewer PCR cycles.were required because the previous TA clone
containing
the KGF-2 gene was used as the starting template for this additional round of
amplification.
= The PCR product contained strategically placed Not I and Sal I
restriction enzyme
sites. This PCR product was cloned with the pCR 2.1-TOPO TA Cloning Kit
(Invitrogen/Lifetechnologies) according to the standard protocol conditions.
49
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The newly cloned KGF-2 cDNA was sequenced, and the sequence was confirmed to
be identical to the KGF-2 cDNA sequence (Genbank accession #U67918).
The KGF-2 cDNA clone was shuttled out of the TA cloning vector by digestion
with
a 5' Not I and a 3' Sal I (ligates to Xho 'restriction enzyme cleavage site)
restriction
enzyme. This fragment was directionally cloned between the K14 promoter and
the &bin
polyA sequences in the pUB-Bsd vector using the Not I and )Tho 1 restriction
enzyme sites.
Example 3
Mammalian Expression Vector Design
This Example presents a mammalian expression vector utilized in the present
inyention. The vector is described in Figure 3 and comprises the following
elements: K14
promoter (2.35kb)/KGF-2 cDNA (627bp)/globin intron & poly(A) (1.165kb)/pUB-
13sd
(4.2451(b).
Example 4
KGF-1 niRNA Expression Diagnostic Screen (RT-PCR)
This Example describes the KGF-2 naRNA expression diagnostic screen utilized
in
'4
the present invention. NIKS cells were transfected using Trans-hi-1(er atino
cyte Transfection
TM
Reagent (Mirus Corp.) and grown in either EpiLife Medium (Cascade Biologics)
or INTIK.S
STRATALIFE medium (Stratatech Corporation). Supernatants were collected for
three
days and used in the development of a direct KGF-2 ELISA Assay. After three
days the
TM
cells were lysed with Trizol Reagent (Invitrogen) for RNA isolation. First
strand cDNA
synthesis was performed using total RNA isolated fonn these transiently
transfected NS
cells. The following primer sequences were utilized:
Fwd 5' -TGCTGTTCTTGGTGTCTTCCG-3' (3T135)(SEQ ID NO:6)
KGF-2 Specific Rev 5'-CAACCAGCACGTTGCCCAGG-3 (ST124)(SEQ BD NO:7)
Globin fragment Specific Oligo d(T) 5'TGTTAGCAATCTGAAGTGGGAGC
GGCCGCGCTT1T1T1-1 TTTTTT1-11 1T-3' (ST112)(SEQ 1D NO: 8)
Next, reverse transciiptase reactions were conducted under the following
conditions:
RNA Priming Reaction- 2.5ug total RNA (Template), 0.5mM dNFP mix,.Oligo dT
(0.5ug
)- Incubate 65 for 5 minutes, on ice 3 minutes. First strand cDNA synthesis
reaction (added
to the RNA Priming Reaction)- lx RT buffer (Promega Corp.), Rnase Out (40U)
= = --- ' CA 02758178 2015-07-08
(Invitrogen), NI-MLV RT (200U) (Promega), 42 degrees for 50 minutes, heat 70
degrees for
15 minutes. Ono microliter (l ul) of R1 reaction template was used for the
subsequent PCR
reaction.
Next, PCR was conducted., Following a denaturation at 95 C for 5 minutes,
samples
were subjected to the following for 35 cycles: Denaturation at 94 C for 30
seconds,
Annealing conditions at 60 C for 30 seconds, Extension at 72 C for I minute. A
final
Extension at 72 C for 7 minutes was followed by a 4 C Hold. The RT-PCR
strategy is
diagrammed in Figure 4.
A DNA vector specific product of 1.1kb was observed along with the specific
product associated with first strand cDNA synthesis (KGF-2 RNA specific
Product) of
approximately 600bp was observed. No KGF-2 RNA specific product was observed
in
either the mock (vector w/o KGF-2 cDNA insert) control plasmid tansfection or
the reverse
transcriptase minus control reaction.
Example 5
KGF-2 Protein Expression Diagnostic Screen (Direct ELISA)
This Example describes the KGF-2 protein expression diagnostic screen used in
the
present invention..
MKS cells were transfected using Trans-It Keratinocyte Transfection Reagent
(Ivinus Corp.) and grown in either EpiLife Medium (Cascade Biologics) Or MKS
medium
(Stratatech Corporation). Supernatants were collected for three days and used
in the
development of a direct KGF-2 EL1SA Assay. The 100u1 supernatants were
incubated in
plate (Nunc Immunoassay plate) over night; at a minimum samples were plated in
duplicate.
TM
The next day, the samples were washed 3X (lx PBS/0.05% Tween-20) 300u1/well;
blocked
plate (lx PBS/1% BSA/5% Sucrose) 300u1/well @rt. for 30 minutes; washed 3X (Ix
PBS/0.05% Tween-20) 300ul/well; incubated with rabbit anti-huKGF-2 Ab
(0.2ug/well)
rt for 2 hours; washed 3X (lx PBS/0.05% Tween-20)300u1/well; incubated with
goat anti-
rabbit HRP (0.8ing/m1) Ab- use at 1:1000 dilution @ rt for 30 minutes; washed
3X (lx
PBS/0.05% Tween-20) 300u1/well; prewamied TMB @ rt 100u1/well for 30 minutes
at
room temperature; added 50u1 of 2N H2S 04; read O.D. 450nm and 620nm;
corrected for
plate imperfections (450nni-620nm):
This experiment demonstrates elevated KGF-2 protein levels are detected in the
supernatants of transiently transfected NIKE cells, when compared to either
mock
transfection (empty vector) or medium alone controls.
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Example 6
Isolation of NIKS cells expressing exogenously introduced
full length human KGF-2 protein.
This Example describes the isolation of NIK.S cells that express KGF-2.
A. Clonal Isolation Strategy-
Vector Construct- Keratin 14 promoter/KGF-2 cDNA/pUb-Bsd plasmid. A DNA
fragment encoding KGF-2 was isolated by PCR and sequenced to verify the
identity and
integrity of the PCR product. The DNA fragment was identical to previously
reported
sequences for KGF-2. The DNA fragment encoding KGF-2 was cloned into a
mammalian
expression vector containing a blasticidin resistant cassette. Blasticidin has
been used to
select for stably transfected keratinocytes, which are subsequently able to
undergo normal
differentiation.
To provide for constitutive expression of KGF-2 in keratinocytes of the basal
epidermal layer, constructs were generated in which expression of KGF-2 is
under the
control of the human keratin-14 (K14) promoter. A 2.3 kb genomic DNA fragment
containing the K14 promoter was amplified and its activity was confirmed by
the ability to
promote luciferase expression from the pGL3 reporter plasmid (Promega) in NIKS
cells.
The 2.3 kb K14 promoter was then cloned into the pUb-bsd vector (Invitrogen).
Subsequently, the KGF-2 coding region was cloned downstream of the K14
promoter and a
DNA fragment containing the rabbit P-globin intron and poly (A) signal was
inserted
downstream of the KGF-2 coding region to complete this mammalian expression
vector
construction.
The structure of the final vector was confirmed by restriction enzyme mapping
and
DNA sequencing. Oligonucleotide primers were synthesized and used to examine
the
expression of this construct in MKS keratinocyte cells using semi-quantitative
RT-PCR
analysis. The primers were designed to span an intron in the rabbit p-globin
fragment, such
that PCR products generated from a spliced RNA template is approximately 500
bp smaller
than the corresponding fragment amplified from genomic DNA.
Transfection- Transit-Keratinocyte (Mirus) transfection reagent was used to
introduce
the KGF-2 vector DNA into monolayer NIKS cell cultures. Twenty-four to forty-
eight
hours post transfection the MKS cells were plated onto a blasticidin feeder
layer of cells
and fed with blasticidin selection medium.
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Selection- NIKS keratinocyte clones were cocultured in the presence of
blasticidin
resistance feeder cells and selected for growth in presence of N1KSTm medium
containing
2.5 ug/ml blasticidin. Only those colonies that continued to grow in the
presence of
blasticidin selection for duration of selection (a minimum of 18 days) were
isolated and
expanded for further characterization.
Clone Isolation- A traditional "Ring cloning" method to isolate blasticidin
resistant
colonies re-plated to individual tissue culture plates (p35 and p100)
containing mouse
fibroblast feeder cells. When these cultures reach 80-90% confluence, the p35
cultures are
harvested for expression analysis and the p100 cultures are used for the
subsequent
expansion phase.
Characterization of Stably-transfected NIKS keratinocytes- Stable NIKS
keratinocyte
colonies that survived the selection scheme therefore are presumed to contain
the K14-
KGF-2 expression construct. To confirm the presence of the KGF-2 transgene,
genomic
DNA was isolated from each clone and amplified with vector specific primers.
This PCR
screen was designed to reconcile products derived from transgene DNA from that
of
potential endogenous KGF-2 DNA products. Multiple clones were obtained using
this
construct and associated selection scheme.
Expansion- The results of expression analysis obtained from the p35 cultures
dictate
which clones will be expanded for further characterization. The p100 plates
from cultures
identified as having positive expression are grown to approximately 50-80%
confluence
then expanded onto several plates containing mouse fibroblast feeder cells.
B. Results
Twenty-nine NIKS clonal isolates that survived drug selection were isolated
and
characterized. Four of the 29 originally identified clones did not survive the
expansion
phase. The remaining 25 clones were successfully expanded and confirmed to
express KGF-
2, at the level of transcription, detennined using RT-PCR. Total RNA isolated
from
previous transient transfections served as positive RT-PCR controls. Negative
controls were
identical reactions run in the absence of reverse transeriptase. The presence
of a KGF-2
transgene present in the genome of any clone yielded an anticipated PCR
product of
approximately 1 Kb in size with the use of a transgene specific primer set.
Clones were
categorized by semi-quantitative expression analysis into categories
representing low,
medium or high expression levels.
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Example 7
KGF-2 RNA and Protein Expression in Monolayer Cultures
This example describes experiments analyzing the expression of KGF-2 in
monolayer cell cultures. Each of the confirmed RT-PCR positive clones were
assayed for
protein expression; this effort resulted in the detection of KGF-2 protein
over expression in
supernatants. Commercially available KGF-2 specific antibodies were used to
investigate
protein levels of secreted KGF-2 protein detected in supernatants. Western
Blot and ELISA
analysis was performed on cell culture supernatants of clones and compared to
native NIKS
cell supernatants. A cell growth assay is being developed to investigate
possible biological
effects of conditioned media from cultured NIKS KGF-2 clones compared to
endogenous
NIKS cell supernatants.
A. RT-PCR
Transgene specific PCR products were semi-quantitatively reported relative to
GAPDH specific products. The transgene specific PCR primer set was designed to
produce
a product utilizing the rabbit P-globin intron sequence region restricted to
the transgene; as
a result this product is easily distinguishable from endogenous KGF-2 product.
Transfected cultures were assayed for mRNA expression levels approximately 24
hours post-transfection. A commercially available RNA isolation kit was used
to isolate
total cellular RNA (Invitrogen, Carlsbad, CA). Total RNA provided a suitable
template for
the subsequent first strand cDNA synthesis (reverse-transcriptase) reaction
followed by the
polymerase chain reaction (RT-PCR). Amplification products are resolved on an
ethidium
bromide stained agarose gel. The anticipated PCR products specific for the
transgene DNA
and mRNA product is 1.0 Kb and 550 bp respectively.
An additional RT-PCR primer set was designed to specifically amplify the KGF-2
gene niRNA product, however this primer set does not distinguish between
endogenous and
transgene messages. Despite the inability to distinguish endogenous mRNA from
transgene
niRNA intensities were semi-quantitatively compared using the endogenous
control samples
(untransfected and tran,sfected with empty vector) as a point of reference.
To compare the level of KGF-2 RNA expressed from the K14-KGF-2 construct with
KGF-2 RNA from the endogenous gene, RT-PCR analysis was performed using
primers
that will amplify KGF-2 RNA regardless of its origin. Under these conditions
endogenous
KGF-2 has not been identified using these RT-PCR conditions, therefore, KGF-2
does not
appear to be expressed in NIKS keratinocytes. To date, no KGF-2 RT-PCR
products from
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non-transfected MKS cell total RNA controls have been identified. The
anticipated 550 bp
fragment is routinely observed in NIKS cells transfected with the KGF-2
transgene. The
KGF-2 expressed from the K14-KGF-2 construct gives rise to the 550 bp RT-PCR
product.
RT-PCR analysis of two K14-KGF-2 clones show that the 550 bp KGF-2 RNA product
is
overexpressed compared to non-detected endogenous KGF-2 levels. No PCR
products
were seen in control reactions in which reverse transcriptase was omitted,
demonstrating
that these products are derived from RNA and not from template contamination
of the PCR
reactions. These results demonstrate that NIKS clones stably-transfected with
the K14-
KGF-2 expression construct specifically overexpress the KGF-2 transgene.
B. Western Blot
Western blot analysis demonstrates specific products at anticipated gel
positions that
correspond to post translational modification forms of KGF-2 reported in the
literature.
Prominent KGF-2 specific protein bands are observed between 19 and 30 kDa.
Specific
KGF-2 band product intensities observed in Western blot analysis con-oborate
the semi-
quantitative RT-PCR expression results. Endogenous KGF-2 is not detected in
unmodified
NIKS control cultures; these findings are consistent with results obtained
from semi-
quantitative mRNA expression analysis. A positive control (recombinant human
KGF-2)
protein was used at concentrations ranging from 0.3 to 0.5ng/lane that
routinely corresponds
with the 191cDa KGF-2 protein band.
To quantify KGF-2 protein expression in stably-transfected K14- KGF-2 clones,
a
KGF-2 Sandwich ELISA (Polyclonal antibodies from R&D Systems and Santa Cruz)
was
developed to compare KGF-2 levels between various K14- KGF-2 clones and
untransfected
NIKS cells. Supernatants from several K14- KGF-2 clones contain elevated
levels of KGF-
2 compared to unmodified NIKS cell control samples. This increase in KGF-2
protein
expression is consistent with the increase seen by RT-PCR analysis. These
results
demonstrate that NLKS cells can be engineered to stably express and secrete
elevated levels
of KGF-2 protein.
C. ELISA
A Sandwich assay was developed to compare secreted KGF-2 levels; assay results
are reported as amount of protein detected per milliliter of cell supernatant.
The level of
KGF-2 protein detected in supernatants is well above levels detected in
unmodified NIKS
cell supernatants (negative control) samples. ELISA values were obtained for
individual
clones and used to assign relative expression levels.
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Taken together, the expression analysis compiled from each of these assays was
used to group clones into relative expression levels when compared to one
another.
Example 8
KGF-2 RNA and Protein Expression in Organotypie Cultures
This example describes experiments analyzing the expression of KGF-2 in
organotypic cultures.
A. RT-PCR- Comparison of biopsy samples (clones versus NTKS)
The expression of KGF-2 mRNA was examined by RT-PCR in skin tissue generated
from stable clones. Total RNA was extracted from skin tissue and subjected to
RT-PCR
using primers that detect mRNA expressed from the KGF-2 transgene, but not
from an
endogenous KGF-2 gene. KGF-2 mRNA was detected in skin tissue prepared from a
K14-
KGF-2 clone, but was not detected in RNA from skin tissue prepared from
untransfected
NIKS cells. These results demonstrate that the K14- KGF-2 construct is
expressed within
the context of stratified epidermis.
B. Western Blot
Results were similar to those obtained for the monolayer cell cultures.
C. ELISA
Results were similar to those obtained for the rnonolayer cell cultures.
D. Histology- Biopsy of clones versus N1KS
To verify that stably-transfected clones containing the K14- KGF-2 expression
constructs undergo normal epidermal differentiation, cultured skin tissue
containing these
clones was prepared. After two weeks in organotypic culture, K14- KGF-2 clones
formed
cultured skin tissue with normal epidermal morphology. These findings indicate
that
elevated expression of KGF-2 does not interfere with the ability of NLKS cells
to undergo
normal epidermal differentiation.
Example 9
Use of Skin Equivalents Expressing Exogenous KGF-2 to Close Wounds
This Example describes preliminary experimental results obtained when skin
equivalents expressing exogenous KGF-2 were used to close wounds in a mouse
wound
model. In this experiment, organotypic cultured skin (i.e., skin equivalents)
were grafted
onto the denuded back of athyrnic nude mice. Skin equivalents containing
native NIKS
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cells were compared to genetically modified skin equivalents expressing KGF-2.
All tissues
were meshed (2:1 ratio) immediately prior to being grafted onto mice.
Interstitial wound
space closure was monitored in the mice. Each observation time point included
recording
micrometer measurements of the wound area; these measurements were
supplemented with
digital photography. At post operative day 3 (POD 3), complete wound closure
of
interstitial spaces have been observed in the mice with the genetically
modified NEKS
organotypic skin tissue (KGF-2), but not observed in mice grafted with the
NIKS culture
tissue control.
Example 10
Mammalian Expression Vector Design
This Example describes a mammalian expression vector utilized in some
embodiments of the present invention. The vector is described in Figure 5 and
comprises
the following elements: Involucrin promoter (3.7kb)/KGF-2 cDNA (627bp)/globin
intron &
poly(A) (1.165kb)/pUB-Bsd (4.245kb).
Construction of Expression Vector
A genomic DNA fragment containing the human involucrin promoter sequence was
isolated using PCR primers based on published sequences (Crish et al,, i Biol
Chem, 1998.
273(46): p. 30460-5). The integrity of the cloned involucrin promoter PCR
product was
confirmed by restriction enzyme analysis and DNA sequencing using involucrin
specific
primers. The involucrin promoter is not expressed in undifferentiated
keratinocytes, but is
specifically activated in differentiated keratinocytes. It is preferable to
direct
overexpression of the KGF-2 to differentiated keratinocytes to avoid
interfering with normal
keratinocyte differentiation.
The coding region for the KGF-2 gene is cloned into the pUB-Bsd expression
vector
(Invitrogen, Carlsbad, CA). This vector is modified by inserting the
involucrin promoter
upstream of the multiple cloning site. This vector contains the blasticidin
drug selection
cassette that utilizes the ubiquitin promoter sequence driving blasticidin
gene expression.
Briefly, gene specific primers for KGF-2 were designed to contain terminal
restriction
enzyme sites (5'-Eco RV and 3'-Spe I). These primers were used in a PCR
reaction
containing TA cloned cDNA template. The modified KGF-2 PCR product (containing
temnnal restriction enzyme sites) was cloned into the TA cloning vector
(Invitrogen) then
sequenced. The KGF-2 cDNA gene product was shuttled from the TA cloning vector
into a
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mammalian expression vector. Complete mammalian expression vector construction
required a two step vector assembly approach shown in Figure 5.
KGF-2 mRNA Expression Diagnostic Screen (RT-PCR)
A mRNA expression screen was perfomed as described in Example 4.
Involucrin promoter/KGF-2 Expression Construct
1) Electroporation transfection method results-
Table 1: Summary of clonal selection and mRNA expression results.
Experiment Clones Picked Clones Survived
Positive
54:29 4 4 4
54:31 2 2 2
68:31 5 3 2
2) Trans-1T keratinoeyte transfection method results-
Table 2: Summary of clonal selection and mRNA expression results.
Experiment - Clones Picked Clones Survived-
Positive
58:51 (TransIT) 16 2 2
90 Isolation of NIKS cells expressing exogenously introduced full length
human
KGF-2 Protein
A. Clonal isolation strategy--
Vector Construct- This clonal isolation strategy includes the use of a DNA
maxiprep
(Qiagen) of the Involucrin/ KGF-2 cDNA/ Globin poly(A) fragment/pUb-Bsd
plasmid.
TransIT-keratinocyte Transfection Method- Transit-Keratinocyte (Mirus)
transfection reagent was used to introduce the KGF-2 vector DNA into monolayer
NIKS
cell cultures. Twenty-four to forty-eight hours post transfection the NIKS
cells were plated
onto a blasticidin feeder layer of cells and fed with blasticidin selection
medium.
Electroporation Transfection Method- Early passage NIKS cells were harvested
at
@ approximately 50-70% confluence. Cells were pelleted and the pellet
resuspended
(2x106 cells/800u1) in F-12/DME (5:1).
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800u1 of MKS cell suspension was placed in 0.4cm electroporation cuvette, DNA
was added (10-3Oug, linear or supercoiled), placed in cuvette holder of the
GenePulser and
started. All steps were done at room temperature; the cells were not placed on
ice at any
time during this procedure. The actual voltage and capacitance values were
recorded
Electroporated NTKS cells were removed from the cuvette and diluted into 25-50
mls of fresh NIKS medium, mixed well by pipetting, and plated (5-10 mls) per
p150
containing blasticidin resistant feeders (using either 5 or 10 p150's per
transfection
reaction).
The following day, the medium is replaced on the p150's with blasticidin
containing
medium (2.5ug/mlblasticidin).
BioRad GenePulser Electroporation Settings:
Exponential Pulse Program
270 volts
950uF
ohms
0.4cm cuvette
Selection- NIKS keratinocyte clones were cocultured in the presence of
blasticidin
resistance feeder cells and selected for growth in presence of NIKS medium
containing 2.5
ughnl blasticidin. Only those colonies that continued to grow in the presence
of blasticidin
selection for duration of selection (a minimum of 18 days) were isolated and
expanded for
further characterization.
Clone Isolation- A traditional "Ring cloning" method to isolate blasticidin
resistant
colonies re-plated to individual tissue culture plates (p35 and p100)
containing mouse
fibroblast feeder cells. When these cultures reach 80-90% confluence, the p35
cultures are
harvested for expression analysis and the p100 cultures are used for the
subsequent
expansion phase.
Characterization of Stably-transfected NIKS keratinocytes- Stable NTKS
keratinocyte colonies that survived the selection scheme therefore are
presumed to contain
the Involucrin-KGF-2 expression construct. To confirm expression of the KGF-2
transgene,
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totoal RNA was isolated from each clone to provide a template for RT-PCR
analysis.
Multiple clones were obtained using this construct and associated selection
scheme.
=
Expansion- The results of expression analysis obtained from the p35 cultures
dictate
which clones will be expanded for further characterization. The p100 plates
from cultures
identified as having positive expression are grown to approximately 50-80%
confluence and
then expanded onto several plates containing mouse fibroblast feeder cells.
B. Results-
TransIT-Kerationcyte method of transfection for clonal selection- Sixteen NIKS
clonal isolates that survived drug selection were isolated and characterized.
Only two of the
16 originally identified clones survived the expansion phase. These two clones
were
successfully expanded and confirmed to express KGF-2, at the level of
transcription,
determined using RT-PCR. Total RNA isolated from previous transient
transfections
served as positive RT-PCR controls. Negative controls were identical reactions
run in the
absence of reverse transcriptase. The presence of a KGF-2 transgene present in
the genome
of any clone yielded an anticipated PCR product of approximately 1 Kb in size
with the use
of a transgene specific primer set. Clones were categorized by semi-
quantitative expression
analysis into categories representing low, medium or high expression levels.
Electroporation method transfection for clonal selection - In one selection
experiment, Four NIKS clonal isolates that survived drug selection were
isolated and
characterized. All four originally identified clones survived the expansion
phase. In a
second experiment, Two NIKS clonal isolates that survived drug selection were
isolated and
characterized. Both originally identified clones survived the expansion phase.
In a third
experiment, Five MKS clonal isolates that survived drug selection were
isolated and
characterized. A11 five originally identified clones survived the expansion
phase.
All clones generated in this series of selection experiments were successfully
expanded and confirmed to express KGF-2, at the level of transcription,
determined using
RT-PCR. Total RNA isolated from previous transient transfections served as
positive RT-
PCR controls. Negative controls were identical reactions run in the absence of
reverse
transcriptase. The presence of a KGF-2 transgene present in the genome of any
clone
yielded an anticipated PCR product of approximately 1 Kb in size with the use
of a
CA 02758178 2015-07-08
transgene specific primer set. Clones were categorized by semi-quantitative
expression
analysis into categories representing low, medium or high expression levels.
. .
Example 11
Expression of Endogenous Human Beta Defensins in NIKS Cells
This example provides an analysis of endogenous human beta defensin (1113D)
expression in MKS cells. Since it was unlmomm if NES cells express hIlDs, RT-
PCR
analysis was perfonued to verify detectable levels of hBD-1, h1313-2, and h_BD-
3 in both
monolayer and organotypic cultures of MKS keratinocytes. Specifically, reverse
transcriptase reactions were performed on both monolayer and organotypic MKS
cell
cultures for each of the human B-defensin genes being studied. Reverse
transcriptase
reactions were performed using total RNA isolated from both N1KS cell
monolayer and
organotypic cultures using an oligonucleotide d(T) primer. One microliter of
RT reaction
template was used in a 20u1 PCR reaction containing gene specific prinaers.
PCR reactions
were conducted as follows- Denaturation at 95 C for 5 minutes, samples were
subjected to
the following for 35 cycles: Denaturation at 94 C for 30 seconds, Annealing
conditions at
58 C for 30 seconds, Extension at 72 C for 30 seconds. A final Extension at 72
C for 7
-minutes was followed by a 4 C Hold. Fifteen microliters of a 20u1 PCR
reaction was
resolved on a 1% agarose gel containing ethidium bromide. The gels were
analyzed for the
anticipated PCR product sizes of 275bp, 205bp & 290bp corresponding to hBD-1,
hl3D-2
laBD-3 respectively.
Intact human skin is reported to express all three human hBDs and their
expression
levels are increased in response to injury and inflammation. To date there
have been no
reports on the expression of hBDs in primary htlMall keratinocytes in
monolayer and only
one report on hBD-2 protein expression in a nontherapeutic product, Matek's
EpiD erm. A
thorough analysis of the RNA expression levels of all three bBDs in both
monolayer and
organotypic cultures of NIKS keratinocytes was conducted. Organotypic culture
of NEKS
keratinocytes results in enhanced levels of all h_13Ds relative to monolayer
culture
conditions, although the magnitude of induction varied among the liBDs. In
monolayers of
N1KS cells the steady state mRNA expression levels of hBD-2 and bBD-3 were
below the
limit of detection. liBD-3, a broad spectrum antimicrobial peptide, was poorly
expressed
even in organotypic culture supporting the notion that overexpression of hBD-3
in MKS
61
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--=
-
keratinocytes will result in enhanced antimicrobial properties cs:pecially in
bioengineered
human sldri tissue generated by organotypic culture techniques.
Example 12 =
Cloning of Human 13eta Defensin
This example describes the cloning of hBD-1, h-BD2, and hBD3 from NIKS cells.
The reverse transcriptase-polymerase chain reaction products described in
Example 1 were
cloned into the TArmcloning vector (Invitrogen) and sequenced to confirm their
genetic
identity. A surmnary of the sequencing results for each of the cloned cDNA
products is as
follows, Human 13-defensin-1 cDNA sequence was coffin-fled to be identical to
Genbank
Accession #1/73945 for hBD-1. Sequence of the human (3-defensin-2 cDNA reveled
a point
mutation at amino acid position 448 (Lys--->Arg) when compared to Genbank
Accession
#AF040 153 for liBD-2. The sequence was amplified, using Pfa proof-reading
polymerase,
and cloned. The sequence was confirmed to be identical to the GenBank
sequence.
The sequence of the human13-defensin-3 cDNA clone was originally found to
contain two point mutations at amino acid positions #57 (Thr-> Met) and
#62(Cys--> Tyr).
Pfu polymerase (proof-reading enzyme) was used to successfully re-amplify the
hBD-3
cDNA which was cloned into the TA cloning vector and sequenced. The sequence
of this
new clone is identical to that reported in the Genbank Accession #AF295370 for
11BD-3.
Example 13
= Construction of Expression Vectors
The example describes the construction of hBD expression vectors. A genomic
DNA fragment containing the hum,an involucrin promoter sequence was isolated
using PCR
primers based on published Sequences, Gish, LE, T.M. Zaim, and R.L. Eckert,
The distal
regulatory region of the human involucrin promoter is required for expression
in epidermis.
J Biol Chem, 1998. 273(46): p. 30460-5. The integrity of the cloned involucrin
promoter
PCR product was confirmed by restriction enzyme analysis and DNA sequencing
using
involucrin specific primers. The involucrin promoter is not expressed in
undifferentiated
keratinocytes, but is specifically activated in differentiated keratinocytes.
In previous
studies, we have demonstrated the use of this involucrin promoter fragment
support
expression in monoIayer cultures of MKS keratinocytes. It is preferable to
direct
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overexpression of the 0-defensins to differentiated keratinocytes to avoid
interfering with
normal keratinocyte differentiation.
The coding region for each of the P-defensin genes is cloned into the pUB-Bsd
expression vector (Invitrogen, Carlsbad, CA). This vector is modified by
inserting the
involucrin promoter upstream of the multiple cloning site. This vector
contains the
blasticidin drug selection cassette that utilizes the ubiquitin promoter
sequence driving
blasticidin gene expression. A restriction enzyrne map of the hBD1 vector is
provided in
Figure 12. Briefly, gene specific primers for hBD-1 were designed to contain
terminal
restriction enzyme sites (5'-Xma I and 3'-Xba I). These primers were used in
an RT-PCR
reaction containing total cellular RNA isolated from NIKS cells. The hBD-1 PCR
product
was cloned into the TA cloning vector (Invitrogen) then sequenced. The
defensin cDNA
gene product was shuttled from the TA cloning vector into a mammalian
expression vector.
Complete mammalian expression vector construction required a two step vector
assembly
approach shown in Figure 13. A similar cloning strategy was used to generate
the hBD-2
and liBD-3 mammalian expression constructs.
Example 14
Expression of Exogenous hBD in NIKS Cells
Purified DNA from each of the Involucrin-P-defensin-UB-Bsd vectors was
introduced into NIKS cells. Specifically, NIKS cells were transfected using
Transit-
Keratinocyte reagent (Mims Corporation), which has been used to efficiently
transfect NIKS
cells. Negative control samples included mock transfected (no DNA) or empty
vector (no p-
defensin) transfected populations of NIKS cells.
inRNA analysis: Transfected cultures were assayed for mRNA expression levels
approximately 24 hrs post-transfection. A commercially available RNA isolation
kit was
used to isolate total cellular RNA (Invitrogen, Carlsbad, CA). Total RNA
provided a
suitable template for the subsequent first strand cDNA synthesis (reverse-
transcriptase)
reaction followed by the polymerase chain reaction (RT-PCR). Amplification
products were
resolved on an ethidium bromide stained agarose gel. The anticipated PCR
products specific
for the transgene DNA and naRNA is as follows- hBD-1 (720 bp and 220 bp), hBD-
2 (700
bp and 200 bp), and hBD-3 (710 bp and 210 bp) respectively.
Results of this experiment confirni the anticipated RT-PCR product sizes, for
each
of the three defensins. Also, as anticipated in the reverse transcriptase
minus control
reactions a single robust signal was detected which corresponds to the
amplification of the
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transgene DNA. No specific PCR products were observed in the mock transfected
control
reactions.
Protein analysis: Culture medium from cells transiently transfected with each
of the
three candidate P-defensin transgenes is assayed for P-defensin peptide
production using an
Enzyme-Linked Irnmunosorbent Assay (ELISA) and Western Blotting assays using
anti-p-
defensin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). A comparison
is made to
endogenous levels from non-transfected MKS cells. A synthetic peptide,
positive control, is
included in these assays. Cell lysates may be required for analysis as p-
defensin protein may
remain associated with the outer membranes of the cells rather than freely
secreted into the
culture medium.
Example 15
Isolation of NIKS Cells Expressing Exogenously Introduced Full Length hBD-1
Protein
This Example describes the isolation of NIKS cells that express hBD-1.
A. Clonal Isolation Strategy
Vector Construct- Involucrin pronzoter/hBD- 1 cDNA/pUb-Bsd plasmid. A DNA
fragment encoding hBD-1 was isolated by PCR and sequenced to verify the
identity and
integrity of the PCR product. The DNA fragment was identical to previously
reported
sequences for hBD-1. The DNA fragment encoding hBD-1 was cloned into a
mammalian
expression vector containing a blasticidin resistant cassette. Blasticidin has
been used to
select for stably transfected keratinocytes, :which are subsequently able to
undergo normal
differentiation.
To provide for constitutive expression of hBD-1 in keratinocytes of the
stratified
epidermal layer, constructs were generated in which expression of hBD-1 is
under the
control of the human Involucrin promoter., A 3.7 kb genomic DNA fragment
containing the
Involucrin promoter was amplified then cloned into the plib-bsd vector
(Invitrogen). The
laBD-1 coding region was cloned downstream of the Involucrin promoter and a
DNA
fragment containing the rabbit p-globin intron and poly (A) signal was
inserted downstream
of the hBD-1 coding region to complete this mammalian expression vector
construction.
The structure of the final vector was confirmed by restriction enzyme mapping
and
DNA sequencing. Oligonucleotide primers were synthesized and used to examine
the
expression of this construct in NIKS keratinocyte cells using semi-
quantitative RT-PCR
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analysis. The primers were designed to 'span an intron in the rabbit f3-g1obin
fragment, such
that PCR products generated from a spliced RNA template are approximately 500
bp
smaller than the corresponding fragment amplified from genomic DNA.
Transfection- Transit-Keratinocyte (Mirus) transfection reagent was used to
introduce
the hBD-1 vector DNA into monolayer N1KS cell cultures. Twenty-four to forty-
eight hours
post transfection the NlKS cells were plated onto a blasticidin feeder layer
of cells and fed
with blasticidin selection medium.
Selection- NTKS keratinocyte clones were coeultured in the presence of
blasticidin
resistance feeder cells and selected for growth in presence of NIKS medium
containing 2.5
ug/ml blasticidin. Only those colonies that continued to grow in the presence
of blasticidin
selection for duration of selection (a minimum of 18 days) were isolated and
expanded for
further characterization.
Clone Isolation- A traditional "Ring cloning" method was used to isolate
blasticidin
resistant colonies re-plated to individual tissue culture plates (p35 and
p100) containing
mouse fibroblast feeder cells. When these cultures reach 80-90% confluence,
the p35
cultures are harvested for expression analysis and the p100 cultures are used
for the
subsequent expansion phase.
Characterization of Stably-transfected NIKS keratinocytes- Stable NlKS
keratinocyte
colonies that survived the selection scheme therefore are presumed to contain
the
Involucrin-hBD-1 expression construct. To confirm the presence of the hBD-1
transgene,
total RNA was isolated from each clone and RT-PCR amplified with transgene
specific
primers. This PCR screen was designed to reconcile products derived from
transgene total
RNA from that of potential endogenous hBD-1 expression products. Multiple
clones were
obtained using this construct and associated selection scheme.
Expansion- The results of expression analysis obtained from the p35 cultures
dictate
which clones were expanded for further characterization. The p100 plates from
cultures
identified as having positive expression were grown to approximately 50-80%
confluence
then expanded onto several plates containing mouse fibroblast feeder cells.
B. Results
Thirty MKS clonal isolates that survived drug selection were isolated and
characterized. Ten of the 30 originally identified clones did not survive the
expansion phase.
The remaining 20 clones were successfully expanded and conlimed to express hBD-
1, at
the level of transcription, determined using RT-PCR. Total RNA isolated from
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transient transfections served as positive RT-PCR controls. Negative controls
were identical
reactions run in the absence of reverse transciiptase. The presence of a hBD-1
transgene
detected in the genome of any clone yielded an anticipated PCR product of
approximately
720 bp in size with the use of a transgene specific primer set. Clones were
categorized by
semi-quantitative expression analysis into categories representing low, medium
or high
expression levels.
NIKS cell expression of exogenously introduced full length hBD-3 protein have
also
been isolated in the same fashion as described above.
Example 16
hBD Activity in NIKS Cells
This Example describes assays for hBD activity. To determine if transient
expression of 0-defensins in NICKS monolayer cultures results in enhanced
bactericidal
activity, a modified in vitro inhibition zone assay is utilized. Hultmark, D.,
et al., Insect
immunity. Attacins, a fanzily of antibacterial proteins _from Tlyalophora
cecropia. Embo J,
1983. 2(4): p. 571-6. Briefly, thin (1mm) agarose plates are seeded with a
microbe of
choice E. co/i, S. aureus, P. aeruginosa, S. pyogenes or C. albicans). The
melted agarose
(1%) contains Luria-Bertani broth with or without supplemented salt. Vogel,
H.J.,
Acetylonzithinase of Escherichia coli: partial purification and sonze
properties. J Biol
Chem, 1956. 218: p. 97-106. The test organism, (-5 x 104 log-phase cells/ml)
is added just
before pouring the plate. Small wells (3mm diameter) are punched in the assay
plates and
loaded with 3u1 of harvested culture mediuni conditioned for at least 24 hours
by
untransfected NIKS, NIKS transiently transfected with the empty expression
construct, or
NIKS transiently expressing each P-defensin . Alternatively, discs are loaded
with 3u1 of
harvested conditioned medium described above and placed on a plate containing
the seeded
microbial lawn. A positive control sample of a synthetic hBD-3 peptide (2-
3Oug/m1) or an
antibiotic such as streptomycin (10Oug/m1) is added to the conditioned medium
and assayed,
along with a negative control (unconditioned medium sample). After overnight
incubation
at 30 C, the inhibition zones are recorded using a ruler and if necessary a
magnifying glass.
The units of activity are read from a standard curve with the zones obtained
by a dilution
series for the synthetic 13-defensin peptide (i.e., hBD-3 synthetic peptide).
Garcia, J.R., et
al., Identification of cz novel, multifunctional beta-defensin (human beta-
defensin 3) with
specific antimicrobial activity. Its interaction with plasma membranes of
Xenopus oocytes
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and the induction of macrophage cheinoattraction. Cell Tissue Res, 2001.
306(2): p. 257-
64. Antimicrobial potency is measured and compared to published Standards (hBD-
3
synthetic peptide or streptomycin). Ideally, the square of the diameter of the
inhibition zone
is proportional to the log of the concentration of an antibacterial factor.
Frohm, M., et al.,
__ Biochemical and antibacterial analysis of human wound and blister fluid.
Eur J Biochem,
1996. 237(1): p. 86-92. This cost effective assay is standardly used as a
measure of
antimicrobial activity, however it provides only semi-quantitative results of
antibacterial
activity.
A minimum inhibitory concentration (MIC) assay is also performed. The smallest
__ amount of conditioned medium from NIKS cells transiently transfected with
each of the
defensin genes required to inhibit the growth of the test organism is
determined. In this
assay a series of culture tubes (or wells of a multi-well plate) containing
bacterial growth
medium with varying concentrations of NIKS conditioned medium is inoculated
with the
test organism. After an incubation period the turbidity is measured and the
MIC is
__ determined. Synthetic antimicrobial f3-defensin peptides are used as
positive controls. The
MIC results are compared to those previously published by others (i.e.,
stimulated
concentration range 15-7Oug/gm tissue or 3.5-16uM. Harder, J., et al., Mucoid
Pseudomonas aeruginosa, TNF-alpha, and IL-1 beta, but not IL-6, induce human
beta-
defensin-2 in respiratoty epithelia. Am J Respir Cell Mol Biol, 2000. 22(6):
p. 714-21.
__ These relative ranges are only intended to provide guidance in an effort to
achieve a
reasonable point of reference.
Example 17
Organotypic Culture
This Example describes assays for hBD expression in organotypically cultured
NIKS cells. Stable genetically-modified NIKS clones that demonstrate greater
than two
fold higher expression levels and enhanced antimicrobial activity over
endogenous f3-
defensin gene expression in NIKS monolayer cultures are candidates of further
characterization efforts. These efforts include preparing organotypic cultures
to assess in
__ vitro skin tissue for nomial tissue morphology. A range of P-defensin
expression levels are
examined because expression levels that are too high may hinder the ability to
obtain
normal tissue morphology.
NIKS cell clones that exhibit several different increased 13-defensin
expression levels
are used to prepare human skin substitute tissues using organotwic culturing
techniques.
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CA 02758178 2011-11-07
See, e.g., U.S. Application 10/087,388; 10/087,346; 10/087,641 and PCT
Application US
02/06088. The organotypic cultures
consist of dermal and epidennal compartments. The dermal compartment is formed
by
=
mixing normal human neonatal fibroblasts with Type I collagen in Ham's F-12
meditun
containing 10% fetal calf serum and penicillin/streptomycin and allowing
contraction. The
epidermal compartment is produced by seeding NIKS cells on the contracted
collagen gel in
25 }11 of a mixture of Ham's F-12:DME (3:1, final calcium concentration 1.88
mM)
supplemented with 0.2% FCS, 0.4 gg/inl hydrocortisone, 8.4 ng/ml cholera
toxin, 5 ug/m1
insulin, 24 jig/ml adenine, and 100 units/ml Pig. Cells are allowed to attach
2 hours at
37 C, 5% CO2 before flooding culture chamber with media (day 0). On day 2
cells are fed
with fresh medium. On day 4, cells are lifted to the air/medium interface on
the surface of a
media-saturated cotton pad, which allows the cultures to be fed from below.
Organotypic
cultures are incubated at 37 C, 5% CO2, 75% humidity and are fed fresh medium
every 2
days. By day 10, the NIKS cells stratify to form the basal, spinous, granular
and cornified
epidermal layers.
Histological sections of skin substitutes :tissuesformed by genetically
modified
NIKS cells are compared to cultures prepared from unmodified NIKS cells.
Tissue sections
are stained with hematoxylin and eosin to visualize the stratified epidemial
layers. Cultures
are examined for tissue morphology. Only those 13-clefensin-expressing clones
that exhibit
normal tissue organization and histology are used.
The organotypic cultures in the initial expression studies are prepared using
cells
expressing individual P-defensin transgenes. However, chimeric organotypic
cultures ca be
prepared by mixing NIKS cells overexpressing different f3-defensins to achieve
a broader
range of antimicrobial activities. The cells expressing P-defensin transgenes
can be used in
conjunction with cells derived from a patient (See, e.g., U.S. Appl.
2002/0192196) or in
conjunction with untransfected MKS cells so that potency can be adjusted. This
strategy
provides further flexibility in protein expression profiles in skin tissue.
Example 18
Analysis of Stable hBD mRNA Expression in Organotypic Cultures
This Example describes assays for hBD mRNA. Total cellular RNA is isolated
from
whole tissue samples. This total RNA is used as a template for the subsequent
first strand
cDNA synthesis (reverse-transcriptase) reaction followed by the polyrnerase
chain reaction
(RT-PCR). Amplification products are resolved on an ethidium bromide stained
agarose gel.
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The anticipated PCR products specific for the transgene DNA and mRNA product
is 1.5 Kb
and 720 bp respectively.
Example 19
Analysis of hBD Protein Expression in Organotypic Cultures
To monitor changes in f3-defensin expression in cultured skin substitute
tissue,
media underlying the cultures are harvested at various times. When organotypic
cultures are
days old, they are incubated for 48 hours in fresh medium. After 48 hours
media is
harvested every 12 hours for four days and the levels of f3-defensin protein
in the media is
10 determined by ELISA and/or Western Blot analysis. A comparison is made
to endogenous
gene expression levels of cultured skin substitute tissues made with
untransfected NIKS
cells. In some experiments, tissue lysates are generated in order to detect f3-
defensin
protein.
Example 20
Antimicrobial Analysis of Stable p-Defensin Clones in Organotypic Cultures
Inhibition Zone Assay of Antimicrobial Activity: To determine if human skin
substitute tissue generated from 13-defensin-expressing MKS cells results in
enhanced
bactericidal activity, a modified in vitro inhibition zone assay is utilized.
Both the
conditioned medium and biopsy punches from 14, 21, and 28 day old skin
substitute tissues
are analyzed for antimicrobial activity. Briefly, thin (lnun) agarose plates
are seeded with a
microbe of choice (E. coli, S. aureus, P. aeruginosa, S. pyogenes or C
albicans). The
melted agarose (1%) contains Luria-Bertani broth with or without supplemented
salt. The
test organism, (-5 x 104 log-phase cells/m1) is added just before pouring the
plate. To assay
for 13-defensin activity in conditioned medium from skin substitute tissues
small wells (3mm
diameter) are punched in the assay plates and loaded with 3u1 of harvested
culture medium
conditioned for at least 24 hours by human skin substitutes generated with
untransfected
NIKS or NIKS clones stably expressing each f3-defensin. Alternatively, discs
may be loaded
with 3u1 of harvested conditioned medium described above and placed on a plate
containing
the seeded microbial lawn. A positive control sample of a synthetic hBD-3
peptide (2-
3Oug/m1) or an antibiotic such as streptomycin (10Oug/m1) will be added to the
conditioned
medium and assayed, along with a negative control (unconditioned medium
sample). To
assay the human skin substitute directly, four 8 min punches are collected
from each 44 cm2
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circular skin substitute tissue. As described above, each biopsy punch is
homogenized
(PowerGen Homogenizer), and placed on a plate containing the seeded microbial
lawn.
After overnight incubation at 30 C, the inhibition zones are recorded using a
ruler and if
necessary a magnifying glass. The units of activity are read from a standard
curve with the
zones obtained by a dilution series for the synthetic P-defensin peptide
(i.e., hBD-3
synthetic peptide). Antimicrobial potency is measured and compared to
published standards
(11BD-3 synthetic peptide or streptomycin). Ideally, the square of the
diameter of the
inhibition zone is proportional to the log of the concentration of an
antibacterial factor.
Frohm, M., et al., Biochemical and antibacterial analysis of human wound and
blisterfluid.
Eur J Biochem, 1996. 237(1): p. 86-92. This cost effective assay is standardly
used as a
measure of antimicrobial activity, however it will provide only semi-
quantitative results of
antibacterial activity.
Micro-broth Dilution Assay: A minimum inhibitory concentration (MIC) assay is
performed. The smallest amount of conditioned medium and biopsy punches from
14, 21,
and 28 day old skin substitutes from NIKS cells stably transfected with each
of the p-
defensin genes required to inhibit the growth of the test organism are
determined. In this
assay, a series of culture tubes (or wells of a multi-well plate) containing
bacterial growth
medium with varying concentrations of conditioned medium from skin substitute
tissues is
inoculated with the test organisni. To assay the human skin substitute
directly, four 8 mm
punches are collected from each 44 cm2 circular skin substitute tissue. As
described above
each biopsy punch is homogenized (PowerGen Homogenizer), and varying
concentrations
are incubated with the test organism. After an incubation period the turbidity
is measured
and the MIC is determined. Synthetic antimicrobial P-defensin peptides are
used as positive
controls. The MIC results are compared to those previously published by others
(i.e.,
stimulated concentration range 15-7Oug/gm tissue or 3.5-16uM. These relative
ranges are
only intended to provide guidance in an effort to achieve a reasonable point
of reference.
Bacterial Growth Assay: To evaluate antimicrobial effects ofI3-defensins on
microbes, cell culture supernatants from stable NIKS clones (either monolayer
or
organotypic cultures) will be evaluated for the ability to inhibit bacterial
growth. Cell
culture supernatants will be inoculated with approximately 4X106 e.f.u. of
bacteria, in
triplicate, and incubated for 1-4 hours at 37 degrees. Cell culture media
supernatant
collected from a native NIKS cell culture (i.e. non-genetically modified) will
serve as an
experimental control. NlKS cell culture supernatants spiked with purified P-
defensin
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peptide titrations will be used as positive controls for antimicrobial
activity. Immediately
following the 1-4 hour incubation period, serial dilutions of each culture
condition will be
plated on-I:II/agar plates and incubated at 37 degrees for 18-20 hours.
Triplicate plates for
each serial dilution are assessed for colony founing units.
Example 21
Expression of Defensins in NIKS cells
This example describes elevated 13-defensin expression levels in transiently
transfected NIKS cell mortolayer cultures. Purified DNA front each of the
Involucrin-ii-
defensin-Ub-Bsd vectors (Figure 14) was introduced into MKS cells using
Transit-
Keratinocyte reagent (Mims Corporation, Madison, WI). Mock transfected (no
DNA) or
empty vector (no 13-defensin) transfected populations of NIKS cells were also
analyzed for
endogenous expression levels.
Characterization of Transient 13-Defensin Transgene Expression in Monolayer
NIKS
Cell Cultures
Expression of (3-defensin mRNA from the involucrin expression constructs was
detected in transiently-transfected NIKS monolayer cell cultures by RT-PCR
(Figure 15).
Primers were designed to amplify only the f3-defensin transgene transcripts
from the
involucrin expression vectors and do not detect endogenous P-defensin
expression mRNA.
Also, to minimize amplification from DNA template, DNase treatment was
performed on
each of the total mRNA samples prior to the first strand cDNA synthesis
(reverse
transcriptase) reaction. 13-defensin specific expression mRNA products
(arrowhead) can be
distinguished from. PCR products amplified from expression vector DNA in that
they lack
the rabbit P-globin intron and are therefore 600 bp smaller than products
amplified from
DNA (see Figure 14).
The ability to achieve expression of each 13-defensin transgene was examined
using
transient transfections. MKS keratinocyte monolayer cells (1x106 per well)
were
transfected with the Involucrin-13-defensin-Ub-Bsd plasmid (101,tg) overnight
using
TransIT-keratinocyte reagent (Mirus Corporation, Madison, W1). A control mock
transfection that contained NIKS cells without the addition of plasmid DNA was
also
included. One day post transfection, cells were collected. Total RNA was
isolated using
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Trizol reagent. (Invitrogen, Carlsbad, CA) and was analyzed by RT-PCR to
monitor 13-
defensin gene expression from each of the Involucrin-p-defensin-Elb-Bsd
constructs.
Results-of the 1T-PCR analysis -of P;defensin gene-expression arc shown in
Figure
15. PCR primers were designed to amplify P-defensin mR_NA expressed frorn the
transgene
construct, but noi endogenous hBDmRNA. These Timers also amplify DNA from the
Involucrin-P-defensin-Ub-Bsd plasmids, but this prodtict can be distinguished
from the
spliced niRNA product because it contains the rabbit p-globin introit and so
is 600 bp larger
than the spliced product (see Figure 14). A prominent PCR product
corresponding to
spliced 13-defensin mRNA (arrowhead) is detected for h.13D-1, hBD-2 and hBD-3
(Figure 15
lanes 1, 5, anti 9 respectively). This product is not seen in control
reactions laelcing reverse =
transcriptase (Figure 15 lanes 3, 7, and 11), demonstrating that it is derived
from mR_NA.
These results also show that each of the hBD expression constructs is
expressed in NIKS
keratinocyte cell cultures. =
Expression of Exogenous ,p-Defensin Protein in NIES Cells
Culture medium from cells transiently transfected with. each of the three P-
clefensin
constructs was assayed for overexpression of protein by immunoblot analysis
using anti-p-
defensin antibodies specific for hBD-1, liBD-2 (Santa Cruz Biotechnology,
Santa Cruz, CA)
and hBD-3 (SAGE BioVentures,Carlsbad, CA).
Conditioned medium and cell lysates from transiently transfected monolayer. of
NEU keratinocyte cultures were analyzed separately by SDS-PAGB under
denaturing,
reducing conditions and the levels of hBD-3 protein assessed by immunoblot
analysis.
Transient transfection of N1KS monolayer cultures was perforrned and one day
post
transfection, monolayer culture supernatants and cell lysates were collected
as previously
described for niRNA expression analysis. A BCA protein assay kit (Pierce,
Rockford, EL)
was used to establish a predetermined amount of protein to be loaded into each
well of a
1 6% Tricine NovexTM pre-cast gel (lnyitrogen, Carlsbad., CA) and then
electroblotted onto a
PVDF (0.2 p.m pore size) filter. After blocking with 4% skim milk in phosphate-
buffered
saline for 1 hour, the filter was incubated overnight with a rabbit polyclonal
antibody
purified against amino acid residues 23-33 of the human P-defensin-3 protein
(1:500). The
filter was then incubated with goat anti-rabbit IgG horseradish peroxidase-
conjugated
secondary antibody (1:5000) for 1 hour. = Products were detected by incubating
blots with
enhanced chemiluminescence (ECL) immunoblotting detection system (Amersham
Pharmacia Biotech, Sunnyvale, CA) and exposing to fihn.
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The anticipated product size of hBD-3 protein is 5 kDa. However recent studies
have reported that hBD-3 protein exhibits a molecular weight of approximately
14 kDa,
consistent with the formation of a dimer (Sehibli, D.J., et al., J Biol Chem,
2002. 277(10): p.
8279-89). This difference in weight may be in part explained by post-
translational
modification of proteins (i.e., glycosylation) or is due to dimers of liBD-3
present as a result
of non-reduced disulfide bonds. Synthetic, control hBD-3 (90ng) was detected
by
immunoblot analysis (Figure 16, lane 1). hBD-3 protein of the expected
molecular weight
(5 kDa or 14 kDa) was not detected in conditioned medium harvested from
transfected or
mock (untransfected) NIKS (see Figure 16, lanes 4 & 5). The presence of the
high
molecular weight band observed in lanes 4 and 5 appears to be dependent on the
presence of
serum in the conditioned medium. Only a very faint high molecular weight band
was
observed in seruni-free conditioned medium harvested from transfected or mock
(untransfected) NIKS keratinocytes.
A14 kDa protein recognized by the anti-h13D-3 antibody in cell lysates from
both
transiently transfected and mock transfected MKS cell lysates was detected
(Figure 16,
lanes 6 and 7). MKS transiently transfected with the hBD-3 transgene produce
increased
levels of hBD-3 protein. These cell lysate results indicate that, although hBD-
3 protein is
over-expressed in transiently-transfected NIKS cells, it remains associated
with the cells or
extracellular matrix and does not appear to be secreted into the medium. The
secretory
signals for the granules that contain sequestered p-defensin peptide appear to
be tightly
associated with late stages of squamous differentiation (Oren, A., et al., Exp
Mol Pathol,
2003, 74(2): p. 180-2).
Example 22
Antimicrobial activity of p-defensin in transiently transfected NIKS cell
monolayer
cultures.
This Example describes antimicrobial activity of defensins in cell culture.
Development of an Antimicrobial Assay Used to Detect Biological p-Defensin
Activity
The antimicrobial activity assay employed Escherichia coli and Staphylococcus
cartzosus and is a modification of the protocol described by Porter and
coworkers (Porter,
E.M., et al., Infect Inunun, 1997. 65(6): p. 2396-401). Briefly, grain-
positive or gram-
negative bacteria are grown overnight. The following day the test organisms
are
subcultured for 2.5 hr and working dilutions of 104 bacteria/m1 for
Escherichia coli or 105
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bacteria/ml of Staphylococcus carnosus in 10mM sodium phosphate (pH 7.4)-1%
TSB are
created. All the reactions mix 50111 of experimental reagent (lysis,
supernatants or purified
protein) with 50 1 of bacterial suspension. These reactions are then
incubated at 37 C for
1.5 hr. The reactions are diluted 100-fold in 10mM sodium phosphate (pH 7.4)-
1% TSB
and plated on TSB plates using a spiral plater (Spiral Biotech, Norwood, MA).
The plates
are then incubated for 12 to 16 hr at 37 C. Colonies on these plates are
counted and the
number of viable bacteria is determined and expressed as colony forming units
per milliliter
(CFU/ml).
Standard Curves for Antimicrobial Activity of Synthetic hBD-1, hBD-2 and hBD-3
Peptides
Standard curves for the antimicrobial activity of hBD-1, hBD-2, and h_BD-3 are
shown in Figure 17. Among the hBD proteins, hBD-3 exhibited the most
antimicrobial
activity with the concentration necessary to kill 50% (LC50) of E. coli at
2.4pg/m1 (Figure
17a). Both hBD-2 and hBD-1 were less potent than liRD-3 against E. coli
(Figure 17b and
c). hBD-2 had an LC50 of 12.2 ilg/ml for E. coli and hBD-1 had an LC50 of 102
i.tg/ml. The
gam-positive bacteria, S. carnosus, appears to be even more sensitive to hBD-3
with an
LC50 of 0.19m/m1 (Figure 17d).
Neither conditioned medium nor cell lysates from monolayer cultures of N1KS
cells
transiently transfected with hBD transgenes or controls exhibited
antimicrobial activity in
the antimicrobial assay. Endogenous expression of hBD-2 and hBD-3 is observed
only in
organotypic cultures of NIKS keratinocytes not monolayer cultures. Therefore,
the
monolayer culture conditions compromise the ability to use transient
expression
experiments to assay for antimicrobial activities of the hBD-2 and hBD-3
proteins.
Although hBD-1 is expressed in monolayer and organotypic cultures of N1KS
keratinocytes, hBD-1 exhibits the lowest antimicrobial activity in test
organisms. In
addition, it is possible that the transient transfection efficiency of NIKS
cells, which is
generally 20-30%, may not be sufficient to support the hBD levels necessary to
exhibit
antimicrobial activity. These findings led us to the generation of clones of
NIKS
keratinocytes stably expressing an hBD transgene and to conducting
antimicrobial activity
assays using organotypic cultures of these NIKS clones.
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Isolation of Stably Transfected NIKS Keratinocytes
Based on the lack of antimicrobial activity observed in the transiently
transfected
NIKS keratinocytes, stably-transfected MKS clones expressing hBD transgenes
were
isolated. The present invention is not limited to a particular mechanism.
Indeed, an
understanding of the mechanism is not necessary to practice the present
invention.
Nonetheless, it is contemplated that higher levels of hBD expression would be
achieved in
clones stably-transfected with the hBD transgenes. In addition, it was
observed that both
endogenous hBD mRNA and protein levels were enhanced by organotypic culture
NIKS
keratinocytes and that stratification and/or late stage differentiation events
associated with
the development of barrier function may be necessary for hBD processing or
secretion.
Transiently transfected NaS keratinocytes cannot be assayed following
organotypic culture
because full stratification and barrier function requires at least 11 days to
develop and
transient expression of hBDs would be exhausted.
Stable clones of NIKS keratinocytes expressing hBD-3 were first generated. hBD-
3
was selected because it demonstrates the most potency against the two test
organisms and
exhibits antimicrobial activity against both grain positive and gram negative
bacteria.
Multiple independent clones expressing the hBD-3 transgene were obtained by
transfecting
NIKS cells and selecting stably-transfected cells using growth medium
containing
blasticidin (2.5 p.g/m1). Elevated f3-defensin mRNA expression was verified
using RT-PCR
analysis of total RNA isolated from each NIKS clonal cell Iine and served as
an initial
screen for relative expression levels between different clones.
NIKS clones stably expressing the hBD-3 transgene have been isolated and
screened. To quantify relative (3-defensin expression levels in stably-
transfected NIKS
Involucrin-Defensin-3-Ub-Bsd clones, total cellular RNA was isolated from
blasticidin-
resistant clones. RT-PCR analysis was performed on all blasticidin-resistant
NIKS clones
transfected with the Involucrin-Defensin-3-Ub-Bsd expression construct.
Conditioned Medium from Organotypic cultures of NIKSTm Clones Stably
Expressing
the hBD-3 Transgene Exhibit Enhanced Antimicrobial Activity
The antimicrobial activity of conditioned medium harvested from organotypic
cultures of the stably-transfected NIKS keratinocyte clone expressing the
highest level of
mRNA was assayed using the method described above. Figure 18 shows that 70%
of the E. coli and up to 52% of the S. carnosus bacteria were killed following
exposure to
conditioned medium from organotypic cultures of NIKS keratinocytes stably
expressing the
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hBD-3 transgene when compared to conditioned medium harvested from organotypic
. cultures generated from untransfected NIKS keratinocytes. Conditioned
medium from
control NIKS organotypic cultures exhibit detectable, but low levels of
antimicrobial
activity, consistent with a link between squamous differentiation and
endogenous hBD-3
expression (Abiko, Y., et al., J Dermatol Sci, 2003. 31(3): p. 225-8.).
Example 23
Defensin Mutants
This example describes site-directed mutagenesis of hBD3. Five (5) of six Cys
were
mutated to Ala (i.e.. Cys40, Cys45, Cys55, Cys62, Cys63). In another mutant,
G1y38 is mutated
to Ala38).
Site-Directed Mutagenesis-
A commercially available kit, QUIKCHANGE Multi Site-Directed Mutagenesis kit
(Stratagene, LaToLla, CA) was used to create amino acid substitutions in the
native hBD-3
polypeptide. The hBD-3 cDNA TopoTA DNA vector was used as the parental DNA
template for the site-directed mutagenesis reactions using the manufacturer
specifications.
Briefly, a therrnocycling reaction included- double stranded DNA template, two
or more
synthetic phosphorylated oligonucleotide primers that contain the desired
mutation(s),
enzyme blend containing PfuTurbo DNA polymerase. First the mutagenic primers
are
annealed to the denatured DNA template. PfuTurbo DNA polymerase was used to
extend
the mutagenic primer(s) generating double stranded DNA molecules with one
strand
bearing the wanted mutation(s). In Step 2, the thermocycling reaction products
were treated
with Dpn I restriction endonuclease. The Dpn I endonuclease is specific for
methylated and
hemimethylated DNA and is used to digest parental DNA template. DNA isolated
from
almost all E. coli strains is darn methylated and therefore susceptible to
this digestion. In
Step 3, the reaction mixture, enriched for mutated single stranded DNA is
transformed into
ultracompetent cells (dam), where the mutant closed circle ss-DNA is converted
to duplex
form in vivo. Double stranded plasmid DNA is prepared from the transfonnants
and clones
are identified that contain the wanted mutation(s).
Synthetic phosphorylated oligonucleotide primers- The mutated codon sequence
is
underlined.
1) G1y38-> Ala mutation oligonucleotide sequence
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(ST262) 5'-Phos-GCA GAG TCA GAG GCG CCC GGT GTG CTG TGC
TCA GC-3' (SEQ ID NO:115)
2) Cys (4o,45,55,69,63)--) Ala mutation oligonucleotide sequences
(ST258) 5'-Phos-CCTCCTTTGGAAGGGCGCTGAGCACAGC
AGCCCGGCCGCC-3' (SEQ ID NO:116)
(ST259) 5'-Phos- CTTTCTTCGGGC GGCTTTTCGGCCACGCGTCGA
GGCCTTGCCGATC-3' (SEQ ID NO:117)
Final mutant amino acid sequences- The site-directed substitutions are
highlighted.
1) Amino Acid Sequence (G1y38-4 Ala)
MRTETYLLFALLFLFLVPVPGHGGENTLQKYYCRVRGARCAVLSCLPKEEQIG
KCSTRGRKCCRRKK (SEQ ID NO:118)
2) Amino Acid Sequence (5Cys--> Ala)
NIRIEIYLLFALLFLFLVPVPGHGGIINTLQKYYCRVRGGRAAVLSALPKEEQI
GICASTRGRICAARRICK (SEQ ID NO:119)
Expression Vector Constructs-
Involucrin promoter hBD-3 mutant cDNA Globin poly(A)
Electroporation Transfection Method- Early passage N1KS cells were harvested
at
@ approximately 50-70% confluence. Cells were pelleted and the pellet
resuspended
(1x106-3x106cells/800u1) in F-12/DME (5:1).
800u1 of NJKS cell suspension was placed in 0.4cm electroporation cuvette, DNA
was added (10-3Oug, linear or supercoiled), placed in cuvette holder of the
GenePulser and
started. All steps were done at room temperature; the cells were not placed on
ice at any
time during this procedure. The actual voltage and capacitance values were
recorded
Electroporated NIKS cells were removed from the cuvette and diluted into 25-50
nals of fresh NIKS medium, mixed well by pipetting, and plated (5-10 rills)
per p150
containing blasticidin resistant feeders (using either 5 or 10 p150's per
transfection
reaction).
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In the next 24-48 hours, the medium is replaced on the p150's with blasticidin
containing medium (2.5ug/mlblasticidin).
BioRad GenePulser Electroporation Settings:
Exponential Pulse Program
270 volts
950uF
ohms
0.4cm cuvette
Selection- NIKS keratinocyte clones were cocultured in the presence of
blasticidin
resistance feeder cells and selected for growth in presence of NIKS medium
containing 2.5
ug/ml blasticidin. Only those colonies that continued to grow in the presence
of blasticidin
selection for duration of selection (a minimum of 18 days) were isolated and
expanded for
further characterization.
Clone Isolation- A traditional "Ring cloning" method is to isolate blasticidin
resistant
colonies. The clones are first picked onto a feeder layer in individual plates
(p60) and
allowed to grow until they are between 80-90% confluent. The clones are then
passed and
re-plated to two individual tissue culture plates (p60 and p100). The p100
contains mouse
fibroblast feeder cells and the p60 does not. When these cultures reach 80-90%
confluence,
the p60 cultures are harvested for expression analysis and the pl 00 cultures
are used for the
subsequent expansion phase.
Characterization of Stably-transfected NIKS keratinocytes- Stable NTKS
keratinocyte
colonies that survived the selection scheme therefore are presumed to contain
the Involucrin
hBD-3 expression construct. To,confirm the presence of the li.BD-3 transgene,
RNA was
isolated from each clone and cDNA products were generated using reverse
transcription
(RT). The RT products were then used as templates in subsequent PCR reactions.
This PCR
screen was designed to reconcile products derived from transgene cDNA from
that of
potential endogenous liBD-3 DNA products. Multiple clones were obtained using
the liBD-
3 constructs (G1y38 Ala substitution and 5 Cys Ala substitution) and
associated
selection scheme.
Expansion- The results of expression analysis obtained from the p60 cultures
dictate
which clones were expanded for further characterization. The p100 plates from
cultures
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identified as having positive expression were grown to approximately 90%
confluence then
harvested and frozen at -80 C in media containing 10% glycerol.
Results are shown below.
Experiment # (Clones picked/ Positive)
Exp 1- Gly--) AIa* 10/10
Exp 2- Gly-> Ala 14/16
Exp 3- Gly--) Ala 11/11
Cys---> Ala** 0/2
Exp 4- Cys--) Ala 2/2
Exp 5- Cys4 Ala 4/4
Exp 6- Cys-> Ala. 3/3 (two more yet to be screened)
Exp 7- Cys4 Ala 25/26 (five clones yet to be screened)
*Mutant construct (G1y4 Ala)-11BD-3 amino acid substation
**Mutant construct (Cys4 Ala)- hBD-3 amino acid substation five of six
cysteines to
alanines.
Example 24
Design and Construction of hCAP18 Expression Vectors
This example describes the design and construction of human cathelicidin
(hCAP18) mammalian expression vectors.
The human cathelicidin (hCAP18) cDNA was cloned from a commercially available
human cDNA library. PCR products were amplified sequenced to confirm the
genetic
identity. This hCAP18 cDNA sequence was confirmed to be identical to that
sequence
deposited in Genbank for hCAP18.
Two hCAP18 mammalian expression vectors were generated. The first vector
contains the
tissue specific kertain-14 promoter, and the second vector utilizes the
involucrin promoter
as an alternative promoter strategy. A linear map and diagnostic digests of
the hCAP18
mammalian expression vectors are shown in Figure 19. The diagnostic
restriction enzyme
digests demonstrate conect banding patterns of the appropriate sizes. Taken
together these
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results account for the overall integrity of the mammalian expression
constructs.
Sequencing across all of the cloning junctions on both final assembled
constructs (K14
hCAP18 and involucrin hCAP18) was also performed to verify the sequence
integrity of
each expression vector.
Expression of hCAP18 from Expression Constructs
RT-PCR analysis was conducted to verify overexpressed levels of hCAP18 from
both expression constructs (Figure 20). Reverse transcriptase reactions were
performed on
monolayer NIKS cell cultures transiently transfected with each of the human
cathelicidin
expression vectors or mock transfected. The anticipated PCR product size of
0.6 kb
corresponding to hCAP18 is shown in transfected cells and as expected this
hCAP18
product is not seen in RNA from Mock transfected monolayer MKS cell cultures.
An
additional set of PCR primers specific for an endogenous house keeping gene
(GAPDH)
was used on the RT reactions to control for RNA integrity and first strand
cDNA synthesis
reactions.
Example 25
hCAP18/LL-37 antimicrobial activity
This example describes the development of an assay to detect LL-37
antimicrobial activity.
In developing this assay a standard kill curve was produced using a
commercially available
LL-37 peptide (Phoenix Pharrnaceuticals, Belmont, CA). The assay is a
modification of the
antimicrobial assay developed to assess biological activity of other
antimicrobial peptides
described above. A standard curve for the antimicrobial activity of LL-37 was
determined
for gram-positive bacteria, S. carnosus using this synthetic peptide. Results
indicated that
LL-37 exhibited potent antimicrobial activity with a concentration necessary
to kill 50%
(LC50) of the S. carnosus at 0.9 ug/ml.
Example 26
=
Electroporation of Cells
This Example describes the use of electroporation to introduce nucleic acids
into
keratinocytes. This Example further describes the use of electroporation to
select for
pluripotent and multipotent cells in a population.
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Protocol:
Harvest early passage NIKS cells @ approximately 50-70% confluence. Pellet
cells
and resuspend NIKS cell pellet (2x106 cells/800u1) in F-12/DME (5:1). This
same protocol
electroporating 1x106NIKS cells in 800u1 with the same success.
Place 800u1 of NIKS cell suspension in 0.4cm electroporation cuvette, add DNA
(10-3Oug, linear or supercoiled) place in cuvette holder of the GenePulser and
push button.
All steps are done at room temperature; the cells are not placed on ice at any
time during
this procedure. Record actual voltage and capacitance values (these values are
indicative of
reproducible electroporation experimental conditions and may be useful for
future
reference).
Electroporated NIKS cells are removed from the Guyette and diluted into 25-50
mls
of fresh NIKS medium, cells are mixed well by pipetting, and plated (5-10 mls)
per p150
containing blasticidin resistant feeders (using either 5 or 10 p150's per
transfection
reaction).
The following day replace medium on the p150's with blasticidin containing
medium (2.5ug/mlblasticidin). Clonal selection of NIKS keratinocytes is
typically carried
out for 18-20 days in blasticidin media (with fresh medium changes every other
day).
The traditional electroporation conditions for mammalian cells as provided by
the
manufacturer (BioRad) are described below. These conditions need to be
optimized; they
are equipment specific and cell type specific.
Electroporation Medium recommended to be minimal or TE at 0.5 - .8 mls.
Cell Density (single cell suspension) 6-8 x 106
Volume of Cells 0.4 ¨ 0.8 mls
DNA 20-200 ug
Gene Pulser (BioRad) Technical Services Recomniended Ranges Using an
Exponential
Protocol.
Gene Pulser Settings
Voltage (V) 200-350
Capacitance (p.F) 500-1000
Resistance ( )
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Cuvette (mm) 0.4
Experiments conducted resulted in the following optimized protocol for
electroporation:
Cell Density (single cell suspension) 1-2 x 106
Volume of Cells* 0.8 mls
DNA** 10-20 ug
* F-12/DME minimal medium (50 mls:10 mls)
**Linear or supercoiled DNA (Qiagen Maxiprep DNA purification)
All steps performed at ambient temperature
Gene Pulser Settings
Voltage (V) 270
Capacitance ( F) 950
Resistance ( )
Cuvette (mm) 0.4
The above protocol was used to select for cells in a populations of cells that
have
stem-cell-like keratinocyte populations. In some embodiments, a drug selection
cassette
was electroporated. In other embodiments, the cells are electroporated in the
absence of any
exogenous nucleic acids. The results are described below.
1. Clonally selected cell population observations ( Drug selection
cassette containing
DNA electroporated and cell populations under drag selection for > 18 days):
1) Selected for keratinocytes having holoclone or meroclone cell morphology-
colony
morphology of tightly packed, uniform cells, smooth colony edges, overall
round
colony morphology.
2) Selected for cells with stem-cell-like properties
3) Selected for cells that exhibit extended proliferative capacity- in
creation of stable
cell lines, these colonies are typically the only surviving colonies after >18
days
under drug selection pressure.
4) Selected for cells with enhanced pluripotency or multipotency.
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5) Colonies without holoclone or meroclone morphology remain smaller and tend
to stop
growing. These colonies do not share the same characteristics as does the
small tightly packed
uniform cells within each large colony. These colonies die-off and most detach
from the plate
during the selection process.
II. Electroporation population observations (Exposed to electroporation
conditions w/o
DNA and not placed under selection):
1) Selected for keratinocytes having holoclone or meroclone cell morphology-
colony
morphology of tightly packed, uniform cells, smooth colony edges, overall
round colony
morphology.
2) Selected for cells with stem-cell-like properties
3) Selected for cells that exhibit extended proliferative capacity-these
colonies are typically the
larger surviving colonies
4) Selected for cells with enhanced pluripotency or multipotency.
5) Colonies without holoclone or meroclone morphology remain smaller and tend
to stop
growing. These colonies do not share the same characteristics as does the
small tightly packed
uniform cells within each large colony.
The results of this experiment demonstrated that populations of cell can be
electroporated with or
without exogenous nucleic acid and cells with the above described properties
are selected for. In
addition, Transgene expression from NIK stable clones obtained using the
electroporation
method of selection have higher expression levels when compared to those
clones obtained using
the Trans-IT keratinocyte (Mirus) transfection method as demonstrated with
semi-quantitative
RT-PCR analysis.
Various modifications and variations of the described method and system of the
invention will be
apparent to those skilled in the art. Although the invention has been
described in connection with
specific preferred embodiments, it should be understood that the invention is
not limited to such
83
CA 02758178 2015-07-08
specific embodiments. Indeed, various modifications of the described modes for
carrying out the
invention that are obvious to those skilled in molecular biology,
biochemistry, or related fields
are intended.
84
CA 02758178 2011-11-07
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