U.S. patent application number 10/025274 was filed with the patent office on 2002-09-12 for methods to enhance wound healing and enhanced wound coverage material.
This patent application is currently assigned to Research Development Foundation. Invention is credited to Barrow, Robert E., Herndon, David N., Perez-Polo, Jose R..
Application Number | 20020128222 10/025274 |
Document ID | / |
Family ID | 22490162 |
Filed Date | 2002-09-12 |
United States Patent
Application |
20020128222 |
Kind Code |
A1 |
Herndon, David N. ; et
al. |
September 12, 2002 |
Methods to enhance wound healing and enhanced wound coverage
material
Abstract
The present invention describes the incorporation of liposomal
gene constructs directly into a wound to further improve wound
repair, or into wound coverage and/or closure materials to enhance
the functionality of the material. The present invention further
describes the use of human fetal membranes (e.g., amnion) enhanced
with the liposomal gene therapy as a wound coverage material in
full-thickness wound repair. The enhanced fetal membranes or
enhanced cadaver skin have advantages over currently used materials
lacking the liposomal gene construct and are an efficient and safe
approach to improve clinical outcome in patients with burn
injuries.
Inventors: |
Herndon, David N.;
(Galveston, TX) ; Perez-Polo, Jose R.; (Galyeston,
TX) ; Barrow, Robert E.; (Galveston, TX) |
Correspondence
Address: |
Benjamin Aaron Adler
Adler & Associates
8011 Candle Lane
Houston
TX
77071
US
|
Assignee: |
Research Development
Foundation
|
Family ID: |
22490162 |
Appl. No.: |
10/025274 |
Filed: |
December 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10025274 |
Dec 19, 2001 |
|
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|
09602183 |
Jun 22, 2000 |
|
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60140196 |
Jun 22, 1999 |
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Current U.S.
Class: |
514/44R ;
424/450 |
Current CPC
Class: |
A61K 38/30 20130101;
A61L 26/0047 20130101; A61K 9/1272 20130101; A61P 43/00 20180101;
A61K 48/00 20130101; A61L 15/32 20130101; A61L 15/40 20130101; A61P
17/02 20180101; A61P 41/00 20180101 |
Class at
Publication: |
514/44 ;
424/450 |
International
Class: |
A61K 048/00; A61K
009/127 |
Claims
What is claimed is:
1. A method of enhancing wound healing in an external wound in an
individual in need of such treatment, comprising the step of:
injecting a cholesterol-containing cationic liposome into said
wound, said liposome comprising at least one gene encoding a growth
factor.
2. The method of claim 1, wherein said wound is selected from the
group consisting of thermal trauma, chemical trauma, excisional
trauma, surgical trauma and abrasion.
3. The method of claim 1, wherein said growth factor is selected
from the group consisting of growth hormone, insulin-like growth
factor-I, keratinocyte growth factor, fibroblast growth factor,
epidermal growth factor, platelet derived growth factor and
transforming growth factor-.beta..
4. The method of claim 3, wherein said growth factor is
insulin-like growth factor-I (IGF-I) and the concentration of said
gene encoding IGF-I in the liposomes is about 2.2 .mu.g/10 .mu.l
liposomes.
5. A method of enhancing wound healing in an external wound,
comprising the step of: covering said wound with a wound coverage
material, wherein said wound coverage material is impregnated with
a cholesterol-containing cationic liposome, said liposome
comprising at least one gene encoding a growth factor.
6. The method of claim 5, wherein said impregnation of said wound
coverage material is performed prior to covering said wound or
subsequent to covering said wound.
7. The method of claim 5, wherein said wound is selected from the
group consisting of thermal trauma, chemical trauma, excisional
trauma, surgical trauma and abrasion.
8. The method of claim 5, wherein said wound coverage material is
selected from the group consisting of human fetal amnion, human
fetal chorion, human cadaver skin and synthetic skin.
9. The method of claim 5, wherein said growth factor is selected
from the group consisting of growth hormone, insulin-like growth
factor-I, keratinocyte growth factor, fibroblast growth factor,
epidermal growth factor, platelet derived growth factor and
transforming growth factor-.beta..
10. The method of claim 9, wherein said growth factor is
insulin-like growth factor-I (IGF-I) and the concentration of said
gene encoding IGF-I in the liposomes is about 2.2 .mu.g/10 .mu.l
liposomes.
11. A method of enhancing wound healing in an external wound,
comprising the step of: covering said wound with a wound closure
material, wherein said wound closure material is impregnated with a
cholesterol-containing cationic liposome, said liposome comprising
at least one gene encoding a growth factor.
12. The method of claim 11, wherein said impregnation of said wound
coverage material is performed prior to covering said wound or
subsequent to covering said wound.
13. The method of claim 11, wherein said wound is selected from the
group consisting of thermal trauma, chemical trauma, excisional
trauma, surgical trauma and abrasion.
14. The method of claim 11, wherein said wound closure material is
selected from the group consisting of human fetal amnion, human
fetal chorion, human syngeneic skin, and human allogeneic skin.
15. The method of claim 11, wherein said growth factor is selected
from the group consisting of growth hormone, insulin-like growth
factor-I, keratinocyte growth factor, fibroblast growth factor,
epidermal growth factor, platelet derived growth factor and
transforming growth factor-.beta..
16. The method of claim 15, wherein said growth factor is
insulin-like growth factor-I (IGF-I) and the concentration of said
gene encoding IGF-I in the liposomes is about 2.2 .mu.g/10 .mu.l
liposomes.
17. An enhanced wound dressing for external wounds, comprising: a
wound coverage material; and a cholesterol-containing cationic
liposome comprising at least one gene encoding a non-insulin-like
growth factor.
18. The enhanced wound dressing of claim 17, wherein said wound
coverage material is selected from the group consisting of human
fetal amnion, human fetal chorion, human cadaver skin, and
synthetic skin.
19. The enhanced wound dressing of claim 17, wherein said growth
factor is selected from the group consisting of growth hormone,
keratinocyte growth factor, fibroblast growth factor, epidermal
growth factor, platelet derived growth factor and transforming
growth factor-.beta..
20. A composition for enhancing wound healing in an external wound,
comprising: a cholesterol-containing cationic liposome, said
liposome comprising at least one gene encoding a non-insulin-like
growth factor; and a pharmaceutically acceptable carrier.
21. The composition of claim 20, wherein said wound is selected
from the group consisting of thermal trauma, chemical trauma,
excisional trauma, surgical trauma and abrasion.
22. The composition of claim 20, wherein said growth factor is
selected from the group consisting of growth hormone, keratinocyte
growth factor, fibroblast growth factor, epidermal growth factor,
platelet derived growth factor and transforming growth
factor-.beta..
23. The composition of claim 20, wherein said composition is
packaged such that said composition can be loaded into a
syringe.
24. The composition of claim 20, wherein said composition is
packaged in a syringe.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional application of U.S. Ser. No.
09/602,183, filed on Jun. 22, 2000, which claims benefit of
priority of provisional application U.S. Serial No. 60/140,196,
filed Jun. 22, 1999, now abandoned.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the field of
trauma medicine and wounds. More specifically the present invention
relates to methods of enhancing wound healing and enhanced wound
coverage materials.
[0004] 2. Description of the Related Art
[0005] Burn injuries represent one of the most severe forms of
trauma. The larger the burn injury, the more severe the
consequences and the higher the chance of poor extended outcomes
and death. There are over 2 million burn patients annually, and
costs for treatment exceed one billion dollars a year. Fire and
burn injuries are the third leading cause of injuries and death in
children aged 1 to 18 years. The number of mortalities from burns
has decreased over the last decade, primarily due to early and
adequate fluid resuscitation, early and aggressive nutritional
support, improved infection control, improved wound care/would
healing, and hormonal modulation.
[0006] Wound healing is of major importance in the recovery of burn
patients, and therefore, their clinical outcome. It has been shown
that early wound excision and tissue grafting improved the
hypermetabolic response and survival after burn injury. Autologous
skin can be used to graft the excised wound (donor-site), however,
this is not an effective treatment in patients with especially
large burns. In these cases, synthetic skin materials or cadaver
skin have been used.
[0007] Wound cover should be distinguished from wound closure.
Wound closure materials are biologically accepted by the wound bed
and become permanently incorporated into the healing wound. On the
other hand, wound coverage materials rely upon incorporation into
the wound coagulum and in-growth of granulation tissues for
adhesion; this phenomenon is characteristic of many wound coverage
materials. Wound coverage materials, in general, do not biodegrade,
and therefore, can only be temporary substitutes for the epidermis.
Wound coverage materials must therefore be replaced with the
patient's skin, either by re-epithelialization or skin grafts. In
the case of temporary coverage, the wound should not be colonized
with bacteria and should be sufficiently superficial that it would
be expected to heal completely within 3 weeks. Epithelial cells
from the epidermal appendages grow and replace the destroyed
epidermis, and gradually, the wound coverage material is shed.
Therefore, the primary goal for the wound coverage materials in
superficial second degree burns are to limit the microbial invasion
of the wound bed (microbial barrier) to thereby prevent infection,
and to limit the access of air to thereby minimize pain.
[0008] Wound coverage materials have also been used for deep
second-degree or third-degree injuries prior to definitive wound
closure with autologous skin in patients with massive burn
injuries. The optimal wound coverage material has yet to be
determined. However, the requirements for wound closure materials
are to mimic normal dermis and epidermis. Specifically, the
requirements of a superior wound closure material are: a) to
provide a nontoxic, antiseptic, noninflammatory, and nonantigenic
barrier to bacteria and other microbes; b) to provide a normal rate
of heat and water conductivity; c) to provide an immediate, uniform
and intimate adherence to the wound bed; d) to provide support for
normal local host defense and wound repair mechanisms; e) to
maintain elasticity and long-term durability; f) to allow growth
potential; and g) to provide long-term mechanical and cosmetic
function with wound contracture properties that are comparable to
split thickness autografts.
[0009] INTEGRA.TM., ALLODERM.TM. or BIOBRANE.TM. demonstrate very
good biocompatibility and healing characteristics. However, these
materials are very expensive, which limits their widespread use.
Cadaver skin is a relatively efficient and cheap approach for wound
coverage. However, the risk of transmission of HIV, CMV, HSV and
hepatitis is a significant concern, and therefore, limits
application of cadaver skin.
[0010] Fetal membranes possess numerous advantageous
characteristics which make this material applicable as wound
coverage material, including: a) low immunogenicity; b) nontoxic,
antiseptic and noninflammatory; c) no HIV, HSV or CMV infection; d)
unlimited quantities (which, therefore, allow fetal membrane tissue
to be an inexpensive alternative to existing skin replacements); e)
variability of length, diameter and thickness; and f) endogenous
mechanical components, such as collagen, laminin and fibronectin,
to thus ensure mechanical stability, growth support and potential
similar to that of normal human skin.
[0011] Biochemically, thermal injury is a particularly severe form
of trauma accompanied by a hypermetabolic response characterized by
high cardiac output, increased oxygen consumption, compromised
immune response and protein and fat catabolism [34]. The burn wound
supports this vulnerable hypermetabolic state by producing and
releasing thromboxane and pro-inflammatory cytokines [35-37]. Wound
healing is thus important to survival and recovery in burn patients
[22, 38-39]. Anabolic agents, such as growth hormone and
insulin-like growth factor-I, have been shown to attenuate the
hypermetabolic response and to improve wound healing [35,
39-41].
[0012] Insulin-like growth factor-I, a small polypeptide
approximately 7.5 kD in size, is an anabolic agent that has been
shown to improve metabolism [35], gut mucosal function [42] and
protein losses [43] after a thermal injury. IGF-I mediates the
actions of growth hormone in the hypermetabolic state by
attenuating lean body mass loss, the compromised immune response,
the acute phase response, and by enhancing wound healing [35, 38,
44-47]. IGF-I treatment improves wound healing by stimulating
collagen formation and the mitogenicity of fibroblasts and
keratinocytes [40, 41, 48]. There are adverse side effects, such as
hypoglycemia, mental status changes, edema, fatigue and headache,
which limit the therapeutic utility of IGF-I in the treatment of
burns [49, 50]. These adverse side effects are most likely due to
supra-physiological doses of free IGF-I, which are required for
biological efficacy [49, 50].
[0013] Selection of an appropriate vehicle for gene delivery is
paramount [1, 2]. Viruses, in particular adenoviruses due to their
specific transfection capabilities, have been used as gene delivery
vehicles [1-3]. Viruses, however, display viral
infection-associated toxicity, immunological compromise, and
possible mutagenic or carcinogenic effects that make this approach
potentially dangerous [1]. Using liposomes as a delivery system
thus becomes an attractive model due to their non-viral
composition, stability, and ability to interact with the cell
membrane [4]. The addition of cationic properties to the standard
liposomal structure and incorporation of cholesterol, together with
the use of cytomegalovirus (CMV) promoters into the cDNA constructs
used for gene transfer, increase the efficacy and levels of
transgenic expression equal to those achieved with adenoviral
constructs [4, 5].
[0014] The prior art is deficient in methods to enhance wound
healing and enhanced wound coverage materials. The present
invention fulfills this long-standing need and desire in the
art.
SUMMARY OF THE INVENTION
[0015] The present invention describes a method of enhanced wound
healing using liposomes carrying genes encoding growth-enhancing
agents. The present invention further describes an enhanced wound
coverage material impregnated with liposomes carrying genes
expressing growth factors to improve wound healing. It is an object
of the present invention to decrease the hypermetabolic response,
and thus, improve the clinical outcome and increase the
survivability after trauma, particularly thermal injury.
[0016] The present invention describes the incorporation of
liposomal gene constructs directly into a wound and/or into the
coverage material to improve wound repair and enhance the
functionality of the wound coverage material. The present invention
further describes the use of enhanced fetal human amnion membrane,
in conjunction with liposomal gene constructs expressing growth
factors, as a transient wound coverage material in full-thickness
wound repair. Fetal membrane has advantages over currently used
materials, such as Integra.TM., Biobrane.TM., Alloderm.TM., and is
an efficient and safe approach to improve clinical outcome.
[0017] One object of the present invention is to provide methods to
enhance wound healing, methods to enhance wound coverage material
and an enhanced wound coverage material.
[0018] In an embodiment of the present invention, there is provided
a method of enhancing wound healing, comprising the step of:
injecting into the wound a liposome, wherein the liposome comprises
at least one gene encoding a growth-enhancing agent.
[0019] In another embodiment of the present invention, there is
provided a method of enhancing wound healing, comprising the steps
of: covering the wound with a wound coverage material, wherein the
wound coverage material is impregnated with a liposome, wherein the
liposome comprises at least one gene encoding a growth-enhancing
agent.
[0020] In yet another embodiment of the present invention, there is
provided a method of enhancing wound healing, comprising the steps
of: covering the wound with a wound closure material, wherein the
wound closure material is impregnated with a liposome, wherein the
liposome comprises at least one gene encoding a growth-enhancing
agent.
[0021] In still yet another embodiment of the present invention,
there is provided an enhanced wound dressing, comprising: a wound
coverage material; and a liposome comprising at least one gene
encoding a growth-enhancing agent.
[0022] In another embodiment of the present invention, there is
provided a composition for enhancing wound healing, comprising: a
liposome, wherein the liposome comprises at least one gene encoding
a growth-enhancing agent; and a pharmaceutically acceptable
carrier.
[0023] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention. These
embodiments are given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The appended drawings have been included herein so that the
above-recited features, advantages and objects of the invention
will become clear and can be understood in detail. These drawings
form a part of the specification. It is to be noted, however, that
the appended drawings illustrate preferred embodiments of the
invention and should not be considered to limit the scope of the
invention.
[0025] FIG. 1 shows a schematic cartoon of the injection sites.
Animals with one injection site received the injection only at
injection site I, whereas animals with 2 injections received the
injections at injection sites I and II. Skin biopsies I, II and III
were taken for analysis 33 days after burn.
[0026] FIG. 2 shows a schematic of the IGF-I plasmid (pcDNA3) cDNA
with the CMV-driven promoter. A structurally similar plasmid for
.beta.-galactosidase (Lac Z gene ) was also encapsulated into the
same liposome.
[0027] FIG. 3 shows percent change in body weights depicted for the
7 day study. * Significant difference between groups at p<0.05.
Data are presented as means.+-.SEM.
[0028] FIG. 4 shows histologic sections of skin after histochemical
reaction for .beta.-galactosidase activity and counterstained with
eosin. (4A). A finely granular blue-green reaction product is
present within many myofibroblastic and histiocytic cells in the
granulation tissue underlying the burn wound. Magnification
.times.380. (4B). Saline-injected (control) dermal tissue
underlying uninjured skin near the burn wound showed no reaction
product. Magnification.times.380.
[0029] FIG. 5 shows the presence of IGF-I mRNA in the skin after
transfection with lipoplexees containing the cDNA coding for IGF-I.
No IGF-I mRNA could be detected in skin biopsies from rats
transfected with liposomes alone or saline (Lanes A and B). There
was a significant amount of IGF-I mRNA in skin biopsies from rats
transfected with IGF-I cDNA (lane C). Representative sample is
shown.
[0030] FIG. 6 shows serum transferrin concentrations during the 7
day study period. Serum transferrin decreased after burn injury.
Liposomes attenuated the drop at 2 and 5 days postburn. *
Significant difference between groups, p<0.05. Data presented as
means.+-.SEM. (Normal serum transferrin: >72 mg/dl).
[0031] FIG. 7 shows serum .alpha..sub.1-acidglycoprotein 1 to 7
days after thermal injury. * Significant difference between saline
and liposomes at p<0.05. Data presented as means.+-.SEM. (Normal
serum .alpha..sub.1-acidglycoprotein: 55-70 pg/ml).
[0032] FIG. 8 shows serum TNF-.alpha. 1 to 7 days after thermal
injury. * Significant difference between saline and liposomes at
p<0.05. Data presented as means.+-.SEM. (Normal serum
TNF-.alpha.: 1-10 pg/ml).
[0033] FIG. 9 shows serum IL-1.beta. 1 to 7 days after thermal
injury. * Significant difference between saline and liposome
administration at p<0.05. Data presented as means.+-.SEM.
(Normal serum IL-1.beta.: 4-20 pg/ml).
[0034] FIG. 10 shows that the area of wound re-epithelization was
measured by planimetry. Rats receiving encapsulated IGF-I cDNA
constructs had the highest per-cent of re-epithelization throughout
the study period compared to the liposomes or saline groups. *
Liposome cDNA vs. Liposome and saline (p<0.05). Data presented
as mean.+-.SEM.
[0035] FIG. 11 shows the area of wound re-epithelization that was
measured by planimetry. Rats receiving multiple injections of
encapsulated IGF-I cDNA constructs had the highest percent of
re-epithelization throughout the study period compared to single
injections. * IGF-I cDNA multiple injections vs. single injections,
p<0.05. Data presented as means.+-.SEM.
[0036] FIG. 12 shows that the presence of .beta.-galactosidase
protein was detected by chemiluminescent reporter gene assay in
skin biopsies I, II and III. (12A) Rats receiving single injection
of the cDNA construct demonstrated a significant decrease in
.beta.-galactosidase expression along the wound edge. * Significant
difference between skin biopsy I vs. III, p<0.05. (12B) Rats
receiving multiple injections demonstrated consistent elevated
levels of .beta.-galactosidase expression. There was no differences
between skin biopsy I, II or III. Data presented as
means.+-.SEM.
[0037] FIG. 13 shows the IGF-I protein concentration in skin
biopsies I, II and III that was measured by RIA. (13A) Rats
receiving a single injection demonstrated a decrease in IGF-I
concentration from biopsy I to III. * Significant difference
between skin biopsy I vs. III, p<0.05. (13B) Animals receiving
multiple injections demonstrated consistent high levels of IGF-I.
Data presented as means.+-.SEM.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention demonstrates the advantages of using
liposomally-delivered vectors expressing growth-enhancing agents to
trauma wounds, particularly thermal injuries, in rats. The present
invention further compares the functionality of various wound
coverage materials, such as fetal membrane, human skin and several
commercially available synthetic materials in Yorkshire
mini-pigs.
[0039] More specifically, the present invention describes the
introduction of liposomal gene constructs coding for
growth-enhancing factors, e.g., insulin-like growth factor-I
(IGF-I), or keratinocyte growth factor (KGF) directly into the
wound bed or into the wound coverage material and compares the
efficacy to non-liposomal-treated wounds. Efficacy of the liposomal
gene therapy is assessed by measuring the take rate and healing
time, examining wound contraction histologically, histology, immune
markers of coverage material rejection and the extent of the
hypermetabolic response at weekly intervals over a 4 month
period.
[0040] Based on the findings reported herein, gene therapy, in
which one or more genes expressing growth factor(s) are delivered
via liposome vehicles injected directly into the wound or injected
into a wound coverage material which is subsequently applied to the
wound will reform and enhance the current treatment for thermal
injuries, skin ulcers and skin grafting procedures.
[0041] The present invention is directed towards methods of
enhancing wound healing and enhanced wound coverage materials.
[0042] The present invention is also directed to a method of
enhancing wound healing, comprising the step of: injecting into the
wound a liposome, wherein the liposome comprises at least one gene
encoding a growth-enhancing agent.
[0043] The present invention is also directed to a method of
enhancing wound healing, comprising the steps of: covering the
wound with a wound coverage material, wherein the wound coverage
material is impregnated with a liposome, wherein the liposome
comprises at least one gene encoding a growth-enhancing agent.
[0044] In yet another aspect, the present invention is also
directed to a method of enhancing wound healing, comprising the
steps of: covering the wound with a wound closure material, wherein
the wound closure material is impregnated with a liposome, wherein
the liposome comprises at least one gene encoding a
growth-enhancing agent.
[0045] In yet another aspect, the present invention is also
directed to an enhanced wound dressing, comprising: a wound
coverage material; and a liposome comprising at least one gene
encoding a growth-enhancing agent. Typically, the liposome
comprising the growth-enhancing agent is delivered to the wound
coverage material by injection, which may be done prior to or
subsequent to applying the wound coverage material to the
wound.
[0046] In yet another aspect, the present invention is also
directed to a composition for enhancing wound healing, comprising:
a liposome, wherein the liposome comprises at least one gene
encoding a growth-enhancing agent; and a pharmaceutically
acceptable carrier. The composition of this embodiment may be
packaged such that the composition can be readily loaded into a
syringe, or alternately, may be packaged directly in a syringe.
[0047] In these embodiments, representative wounds which may be
treated using the compositions and methods of the present invention
include thermal trauma, chemical trauma, excisional trauma,
surgical trauma or abrasion. Preferable liposomes are
cholesterol-containing cationic liposomes. Generally,
growth-enhancing agents are growth hormone, insulin-like growth
factor-I, keratinocyte growth factor, fibroblast growth factor,
epidermal growth factor, platelet derived growth factor or
transforming growth factor-.beta.. Preferably, when the
growth-enhancing agent is insulin-like growth factor-I (IGF-I), and
the concentration of the gene encoding the IGF-I in the liposome is
about 2.2 .mu.g/ml liposome.
[0048] Representative wound coverage materials include human fetal
amnion, human fetal chorion, human cadaver skin, synthetic skin,
and other materials known to those having ordinary skill in this
art. Representative wound closure materials include human fetal
amnion, human fetal chorion, human syngeneic skin, human allogeneic
skin, and other materials known to those having ordinary skill in
this art.
[0049] In accordance with the present invention, there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, "Molecular Cloning: A Laboratory
Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and
II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait
ed. 1984); "Nucleic Acid Hybridization" [B. D. Hames & S. J.
Higgins eds. (1985)]; "Transcription and Translation" [B. D. Hames
& S. J. Higgins eds. (1984)]; "Animal Cell Culture" [R. I.
Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press,
(1986)]; B. Perbal, "A Practical Guide To Molecular Cloning"
(1984). Therefore, if appearing herein, the following terms shall
have the definitions set out below.
[0050] A "DNA molecule" refers to the polymeric form of
deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in
its either single stranded form, or a double-stranded helix. This
term refers only to the primary and secondary structure of the
molecule, and does not limit it to any particular tertiary forms.
Thus, this term includes double-stranded DNA found, inter alia, in
linear DNA molecules (e.g., restriction fragments), viruses,
plasmids, and chromosomes. In discussing the structure herein
according to the normal convention of giving only the sequence in
the 5' to 3' direction along the nontranscribed strand of DNA
(i.e., the strand having a sequence homologous to the mRNA).
[0051] A "vector" is a replicon, such as plasmid, phage or cosmid,
to which another DNA segment may be attached so as to bring about
the replication of the attached segment. A "replicon" is any
genetic element (e.g., plasmid, chromosome, virus) that functions
as an autonomous unit of DNA replication in vivo; i.e., capable of
replication under its own control. An "origin of replication"
refers to those DNA sequences that participate in DNA synthesis. An
"expression control sequence" is a DNA sequence that controls and
regulates the transcription and translation of another DNA
sequence. A coding sequence is "operably linked" and "under the
control" of transcriptional and translational control sequences in
a cell when RNA polymerase transcribes the coding sequence into
mRNA, which is then translated into the protein encoded by the
coding sequence.
[0052] In general, expression vectors containing promoter sequences
which facilitate the efficient transcription and translation of the
inserted DNA fragment are used in connection with the host. The
expression vector typically contains an origin of replication,
promoter(s), terminator(s), as well as specific genes which are
capable of providing phenotypic selection in transformed cells. The
transformed hosts can be fermented and cultured according to means
known in the art to achieve optimal cell growth.
[0053] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide in vivo when
placed under the control of appropriate regulatory sequences. The
boundaries of the coding sequence are determined by a start codon
at the 5' (amino) terminus and a translation stop codon at the 3'
(carboxyl) terminus. A coding sequence can include, but is not
limited to, prokaryotic sequences, cDNA from eukaryotic mRNA,
genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and
even synthetic DNA sequences. A polyadenylation signal and
transcription termination sequence will usually be located 3' to
the coding sequence. A "cDNA" is defined as copy-DNA or
complementary-DNA, and is a product of a reverse transcription
reaction from an mRNA transcript. An "exon" is an expressed
sequence transcribed from the gene locus, whereas an "intron" is a
non-expressed sequence that is from the gene locus.
[0054] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, polyadenylation
signals, terminators, and the like, that provide for the expression
of a coding sequence in a host cell. A "cis-element" is a
nucleotide sequence, also termed a "consensus sequence" or "motif",
that interacts with other proteins which can upregulate or
downregulate expression of a specicif gene locus. A "signal
sequence" can also be included with the coding sequence. This
sequence encodes a signal peptide, N-terminal to the polypeptide,
that communicates to the host cell and directs the polypeptide to
the appropriate cellular location. Signal sequences can be found
associated with a variety of proteins native to prokaryotes and
eukaryotes.
[0055] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site, as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase. Eukaryotic promoters often, but not always, contain
"TATA" boxes and "CAT" boxes. Prokaryotic promoters contain
Shine-Dalgarno sequences in addition to the -10 and -35 consensus
sequences.
[0056] In general, "gene" is intended to include promoter and
regulatory elements operably linked to a coding sequence. Those
promoter sequences and regulatory elements may be the gene's
natural promoter and/or regulatory elements, or they may be
heterologous. A "heterologous" region is an identifiable segment of
DNA within a larger DNA molecule that is not found in association
with the larger molecule in nature. Thus, when the heterologous
region encodes a mammalian gene, the gene will usually be flanked
(e.g., promoter sequences and/or regulatory elements) by DNA that
does not flank the mammalian genomic DNA in the genome of the
source organism. In another example, the coding sequence is a
construct where the coding sequence itself is not found in nature
(e.g., a cDNA in which the genomic coding sequence contains
introns, or synthetic sequences having codons different than the
native gene). Allelic variations or naturally-occurring mutational
events do not give rise to a heterologous region of DNA as defined
herein.
[0057] "Recombinant DNA technology" refers to techniques for
uniting two heterologous DNA molecules, usually as a result of in
vitro ligation of DNAs from different organisms. Recombinant DNA
molecules are commonly produced by experiments in genetic
engineering. Synonymous terms include "gene splicing", "molecular
cloning" and "genetic engineering". The product of these
manipulations results in a "recombinant" or "recombinant
molecule".
[0058] A cell has been "transformed" or "transfected" with
exogenous or heterologous DNA when such DNA has been introduced
inside the cell. The transforming DNA may or may not be integrated
(covalently linked) into the genome of the cell. In prokaryotes,
yeast, and mammalian cells for example, the transforming DNA may be
maintained on an episomal element such as a vector or plasmid. With
respect to eukaryotic cells, a stably transformed cell is one in
which the transforming DNA has become integrated into a chromosome
so that it is inherited by daughter cells through chromosome
replication. This stability is demonstrated by the ability of the
eukaryotic cell to establish cell lines or clones comprised of a
population of daughter cells containing the transforming DNA. A
"clone" is a population of cells derived from a single cell or
ancestor by mitosis. A "cell line" is a clone of a primary cell
that is capable of stable growth in vitro for many generations. An
organism, such as a plant or animal, that has been transformed with
exogenous DNA is termed "transgenic".
[0059] As used herein, the term "amnion" refers to an
extraembryonic thin membrane surrounding the embryo and derived
from ectoderm and mesoderm tissues.
[0060] As used herein, the term "chorion" refers to the outermost
extraembryonic membrane which eventually becomes part of the
placenta, where in addition to respiratory functions, it supplies
nutrients and removes waste.
[0061] As used herein, the term "liposome" refers to a small
vesicle bounded by a bilayer lipid membrane made artificially from
phospholipids. DNA, proteins and other materials can be enclosed
within the liposome and introduced into animal cells by fusion with
the plasma membrane. As used herein, the term "cholesterol-cationic
liposome" refers specifically to a cholesterol-containing
liposome.
[0062] As used herein, the term "growth-enhancing agent" refers to
compounds that promote growth.
[0063] As used herein, the term "take rate" refers to the `healthy
rate`, or `acceptance rate` of the donor tissue onto the host
tissue.
[0064] It is specifically contemplated that pharmaceutical
compositions may be prepared using the liposomes described in the
present invention. In such a case, the pharmaceutical composition
comprises a gene encoding a growth factor, the liposome of the
present invention and a pharmaceutically acceptable carrier. A
person having ordinary skill in this art would readily be able to
determine, without undue experimentation, the appropriate dosages
and routes of administration of the liposome of the present
invention. When used on a patient in therapy, the liposome vehicle
described in the present invention is administered to the patient
or an animal in therapeutically effective amounts, i.e., amounts
that effectively deliver appropriate amounts of the DNA encoding a
growth-enhancing agent. It will normally be administered in
injectable form to the wound or wound coverage material, but other
routes of administration will be used as appropriate. The dose and
dosage regimen will depend upon the nature of the wound (the
severity of the tissue damage) and its size, the patient's history
and other factors. The amount of liposome administered will
typically be based upon wound area, with liposomal injections
approximately 4 cm apart. The schedule will be continued to
optimize effectiveness while balanced against negative effects of
treatment. See Remington's Pharmaceutical Science, 17th Ed. (1990)
Mark Publishing Co., Easton, Pa.; and Goodman and Gilman's: The
Pharmacological Basis of Therapeutics 8th Ed (1990) Pergamon Press;
which are incorporated herein by reference. For local
administration, the liposomes will most typically be formulated in
a unit dosage injectable form (solution, suspension, emulsion) in
association with a pharmaceutically acceptable carrier. Such
carrier are preferably non-toxic and non-therapeutic. Examples of
such carrier are water, saline, Ringer's solution, dextrose
solution, and 5% human serum albumin. Nonaqueous carriers such as
fixed oils and ethyl oleate may also be used. The carrier may
contain minor amounts of additives such as substances that enhance
isotonicity and chemical stability, e.g., buffers and
preservatives. The liposomes will typically be formulated in such
carriers at concentrations of about 1 .mu.g to 10 .mu.g.
[0065] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion:
EXAMPLE 1
Experimental Animals--Rats
[0066] Adult male Sprague-Dawley rats (350-375 g) were placed in
wire bottom cages and housed in a temperature-controlled room with
a 12 hour light-dark cycle. The animals were acclimatized to their
environment for 7 days prior to the start of the blinded study. All
received equal amounts of a liquid diet of Sustacal (Mead Johnson
Nutritionals, Evansville, Ind., USA) and water ad libitum
throughout the study. Each rat received a 60% total body surface
area (TBSA) full-thickness scald burn. Thermally injured rats were
then randomly divided into:
[0067] (a) 2 groups to receive injections of cholesterol-containing
cationic liposomes (20 .mu.l liposomes in 180 .mu.l saline, n=28),
or saline (control, 200 .mu.l, n=28);
[0068] (b) 2 groups to receive weekly subcutaneous injections of
liposomes (10 .mu.l liposomes in 180 .mu.l saline) containing 2.2
.mu.g of an IGF-I cDNA construct and 0.2 .mu.g of the reporter gene
.beta.-galactosidase, Lac Z cDNA construct driven by a CMV promoter
(n=12) at one injection site on the edge of the burn wound (FIG.
1), or weekly subcutaneous injections of liposomes (10 .mu.l
liposomes in 180 .mu.l saline) containing 2.2 .mu.g cytomegalovirus
driven IGF-I cDNA construct and 0.2 .mu.g of the reporter gene for
.beta.-galactosidase, Lac Z cDNA (n=12) at two injection sites on
the edge of the burn wound (FIG. 1); or
[0069] (c) 3 groups to receive weekly subcutaneous injections of
saline (200 .mu.l normal saline; n=10); weekly subcutaneous
injections of liposomes (10 .mu.l liposomes in 180 .mu.l saline)
containing 0.2 .mu.g of the reporter gene for .beta.-galactosidase
LacZ cDNA construct (n=10); or weekly subcutaneous injections of
liposomes (10 .mu.l liposomes in 180 .mu.l saline) containing 2.2
.mu.g of an IGF-I cDNA construct and 0.2 .mu.g of the reporter gene
.beta.-galactosidase LacZ cDNA construct (n=10).
EXAMPLE 2
Experimental Animals--Pigs
[0070] The wound repair mechanisms in Yorkshire mini-pigs are
closest to the same mechanisms in humans. Thus, Yorkshire mini-pigs
are a well-described model and have been used in several
experimental trauma studies. Each of 10 Yorkshire swines receive 4
full-thickness wounds (standard model) under anesthesia and
analgesia. Each wound site is square or rectangular in shape, with
an approximate dimension of 8.times.8 cm, spaced approximately 5 cm
between sites and confined to the area of the upper flank and back.
The wounds must be positioned and placed according to a particular
scheme such that the animal can lie comfortably on its side
following release from the restraining hammock.
[0071] Immediately after wound induction, the wound is covered with
human fetal chorion/amnion, INTEGRA.TM. (Life Science),
ALLODERM.TM. (Life-Cell) or BIOBRANE.TM. (Dow-Hickhan). The cover
is stapled to the unburned wound and covered with triple-antibiotic
ointment-impregnated gauze and a bulky pressure dressing. Staples
and/or sutures are used as required.
[0072] Following surgery, the study animals are suspended in a
hammock restraint while recovering from anesthesia so as to protect
graft sites from injury or dislodgement. The animals remain in this
hammock restraint for no more than 24 hours.
EXAMPLE 3
Liposomes
[0073] The liposomes used were cholesterol-containing cationic
liposomes, DMRIE-C Reagent
(1,2-dimyristyloxypropyl-3-dimethyl-hydroxyl ethyl ammonium
bromide) prepared with cholesterol membrane-filtered water (Life
Technologies, Rockville, Md.). The IGF-I cDNA construct (FIG. 2)
consisted of a cytomegalovirus driven IGF-I cDNA plasmid prepared
at the UTMB Sealy Center for Molecular Science Recombinant DNA Core
Facility (the IGF-I cDNA was a kind gift of G. Rotwein, NIH,
Bethesda, Md.). The doses used were 10% liposomes (the highest
concentration used in DNA transfer experiments that does not have
deleterious consequences on DNA solubility and is compatible with
gene transfer paradigms). In order to determine the time course of
liposomal effects, liposomes were injected intravenously into the
tail vein of rats in group (a) 0.5 h after the thermal injury and
changes were examined over a 7-day period. The volume of 200 .mu.l
is an amount that can be administered into the tail vein of a rat
without causing deleterious side effects, such as cardiac arrest.
Immediately after the thermal injury, each rat in group (b) or (c)
received 0.2 ml of the solutions injected at either one site, 1 cm
from the wound margin, or at two sites distal to each other (FIG.
1). This protocol was repeated once a week for 4 weeks. Lipoplexes
were prepared fresh every week prior to injections.
EXAMPLE 4
Protocol
[0074] Body weights were measured at the same time each day. The
percent change in total body weight generally increased during the
first 2 days followed by a decrease in body weight 2 days postburn.
Rats receiving liposomes maintained their body weight better
compared to the saline-treated controls, p<0.05 (FIG. 3). Rats
were sacrificed by decapitation 1, 2, 5 or 7 days after burn (for
rats in group (a)), or alternatively, 5 days after the last
injection (33 days after burn) for rats in groups (b) and (c).
Blood was collected into serum and plasma separators, spun at 1000
g for 15 minutes, decanted, and frozen at -73.degree. C. Liver and
gastrocnemius muscle were harvested, weighed, sectioned and samples
of each dried at 60.degree. C. to a constant weight. The dry/wet
weight ratios were used to estimate protein content. Three dorsal
skin samples, defined as biopsy I, II or III, were harvested, snap
frozen in liquid nitrogen and stored at -73.degree. C. for analysis
(FIG. 1). There were no differences in dry/wet weight ratios
between liposome-treated and control animals for gastrocnemius
muscle and liver.
[0075] Serum cholesterol, free fatty acids, serum acute phase
proteins (haptoglobin and .alpha..sub.2-macroglobulin),
constitutive hepatic proteins (total protein, transferrin and
albumin), and glucose were measured on a Behring nephelometer
(Behring, Dearfield, Ill.). Serum .alpha..sub.1-acid glycoprotein
was determined by ELISA (Wako Chemicals Inc., Richmond, Va.). A
standard curve for rat .alpha..sub.1-acid glycoprotein
concentrations was linear from 0 to 1500 pg/ml on a logarithmic
scale. Plasma TNF-.alpha. levels were determined by ELISA (Endogen,
Woburn, Mass.). The standard curve used for the quantification of
rat TNF-.alpha. was linear from 0 to 833 pg/ml on a logarithmic
scale. Serum IL-1.beta. levels were determined by ELISA (Biosource
Int., Camarillo, Calif.) and its standard curve used for the
quantification of rat IL-1.beta. was linear from 0 to 1500 pg/ml on
a logarithmic scale. Serum IL-6 was determined by bioassay using
log phase B9 cells (mouse hybridoma line) treated with increasing
serum concentrations. Cell proliferation in response to additional
serum was measured by quantitative MTS reduction
spectrophotometrically.
EXAMPLE 5
Transfection
[0076] Transfection was determined by measuring the presence of
.beta.-galactosidase. .beta.-galactosidase protein was detected by
histochemical staining with Bluo-gal in the three skin biopsies.
Skin specimens were fixed overnight at 4.degree. C. in fixative
consisting of 4% paraformaldehyde in a HEPES-buffered Hanks
solution at pH 7.6. After washing in buffer and phosphate-buffered
saline (PBS), the specimens were incubated overnight at 37.degree.
C. in a 0.1% solution of Bluo-Gal substrate (halogenated
indolyl-.beta.-D-galactoside) (LIFE Technologies, Gaithersburg,
Md., USA), buffered to pH 7.6. After extensive washing, tissues
were embedded in paraffin, and histologic sections made and stained
with hematoxylin and eosin or with eosin alone.
[0077] The presence of .beta.-galactosidase protein was also
detected by chemiluminescent reporter gene assay (GALACTO-LIGHT
PLUS.TM., Tropix Inc. Bedford, Mass. USA) in skin. Samples were
prepared as follows [55]: 100 mg tissue was homogenized in 200
.mu.l of Lysis buffer (40 mM Tris (pH 7.5), 1 mM EDTA, 150 mM NaCl)
for approximately 30 seconds. Samples were centrifuged
(12,000.times.g) for 3 minutes. The supernatant was removed, the
volume measured and stored on ice. The residual pellet was rinsed
with 200 .mu.l lysis buffer and micro-centrifuged. The assay
followed manufacturers directions in 96 well plates.
[0078] The fine granular blue-green reaction product of the
.beta.-galactosidase reaction was predominantly present in the
granulation tissue, composed of spindle-shaped myofibroblasts,
macrophages, and growing small blood vessels underlying the wound.
The cells consistently staining for .beta.-galactosidase were
myofibroblasts, endothelial cells, and macrophages in the areas of
inflammation and included multinucleate giant cells, indicating
preferential transfection of cells with higher proliferation rates
(FIG. 4A). Although most staining for .beta.-galactosidase was in
the cytoplasm, some reaction products were observed outside the
cell boundaries, possibly due to enzymes released by dead cells or
the simple diffusion of reaction products. A small amount of
reaction product was also detected in the matrix of hair follicles
near the injection sits. No .beta.-galactosidase was detected in
saline-treated animals (FIG. 4B).
[0079] .beta.-galactosidase protein expression was increased around
the wound perimeter in rats receiving liposome-encapsulated LacZ
cDNA plus IGF-I cDNA constructs compared to saline treated rats
(p<0.05; Table 1). There was no difference amoung groups in
.beta.-galactosidase concentrations in blood cells, liver, spleen
or kidney. This finding is consistent with the conclusion that
systemic cells were not transfected after subcutaneous cDNA
injections (Table 2).
1TABLE 1 .beta.-galactosidase expression in skin
.beta.-galactosidase (counts/second/ml extract) Site of Saline
Liposome IGF-I cDNA skin Biopsy (n = 10) (n = 10) (n = 10) I 2503
.+-. 366* 5568 .+-. 381 5620 .+-. 509 II 2858 .+-. 556* 5267 .+-.
325 6555 .+-. 811 III 2982 .+-. 664* 6023 .+-. 800 7357 .+-. 1042
Data presented are mean .+-. SEM. *Significant differences vs.
liposomes and IGF-I cDNA (p < 0.05).
[0080]
2TABLE 2 .beta.-galactosidase expression in blood, liver kidney and
spleen .beta.-galactosidase (counts/second/ml extract) Saline
Liposomes IGF-I cDNA Tissue (n = 10) (n = 10) (n = 10) Blood 349
.+-. 37 235 .+-. 48 244 .+-. 116 Liver 1757 .+-. 139 1881 .+-. 956
1620 .+-. 886 Kidney 964 .+-. 90 896 .+-. 100 916 .+-. 98 Spleen
1154 .+-. 217 1460 .+-. 348 886 .+-. 379 Data presented as mean
.+-. SEM.
EXAMPLE 6
IGF-I
[0081] In rats receiving the IGF-I cDNA construct, there was
evidence of IGF-I mRNA in the skin proximal to transfection sites
but not in control or sham-treated tissues (FIG. 5).
[0082] IGF-I protein concentrations were measured by RIA in the
three skin biopsies (FIG. 1). Proteins were extracted by
pulverizing approximately 40 mg of tissue under liquid nitrogen,
adding extraction buffer (PBS.sup.-, 0.25 ml PMSF, 50 mg leupeptin,
100 mg aprotinin, and 50 mg antipain) in a volume 1:7 (7 ml
buffer/gram tissue) and homogenizing the mixture. To allow proteins
to recover, samples were frozen overnight at -80.degree. C. After
thawing, 50 .mu.l of the homogenate was added to 150 .mu.l of
extraction solution and centrifuged at 13,500 rpm for 5 minutes.
100 .mu.l of supernatant was added to 400 .mu.l of neutralization
solution, and the RIA performed as described in the kit guidelines
(Diagnostic System Laboratories, Webster, Tex., USA).
[0083] In all skin biopsies taken, animals treated with the IGF-I
cDNA constructs had higher IGF-I protein concentrations around the
wound perimeter compared to biopsies from animals treated with
liposomes alone or saline (p<0.05; Table 3). There was no
difference amoung groups in IGF-I protein concentration in serum,
liver, spleen or kidney (Table 4).
3TABLE 3 IGF-I protein concentration in skin IGF-I protein (ng/ml)
Site of Saline Liposomes IGF-I cDNA skin biopsy (n = 10) (n = 10)
(n = 10) I 94 .+-. 5 118 .+-. 10 174 .+-. 12* II 96 .+-. 11 113
.+-. 11 171 .+-. 13* III 98 .+-. 6 100 .+-. 5 178 .+-. 14* Data
presented as mean .+-. SEM. *Significant difference vs. Liposomes
and saline, p < 0.05.
[0084]
4TABLE 4 IGF-I protein concentration in blood, liver, kidney and
spleen IGF-I protein (ng/ml) Saline Liposomes IGF-I cDNA Tissue (n
= 10) (n = 10) (n = 10) Blood 139 .+-. 2 132 .+-. 5 135 .+-. 5
Liver 177 .+-. 3 181 .+-. 6 172 .+-. 6 Kidney 188 .+-. 4 186 .+-.
12 188 .+-. 8 Spleen 160 .+-. 3 150 .+-. 7 156 .+-. 6 Data
presented as mean .+-. SEM.
EXAMPLE 7
Constitutive Hepatic Proteins, Acute Phase Proteins and
Cytokines
[0085] Serum total protein concentration decreased after burn
injury. Rats receiving liposomes showed an increase in serum total
protein 5 days after burn (liposomes: 5.6.+-.0.1 g/dl vs. controls:
5.2.+-.0.1 g/dl), p<0.05. Serum transferrin decreased after burn
injury by nearly 30% below normal. Rats treated with liposome
showed an increase in serum transferrin at 2 and 5 days postburn
compared to controls (p<0.05; FIG. 6). There were no significant
differences in serum albumin between the treated and control
groups. Furthermore, no significant differences between treated and
control animals in serum cholesterol, free fatty acid and glucose
could be demonstrated throughout the study period.
[0086] Type I acute phase proteins, serum haptoglobin and
.alpha..sub.1-acid glycoprotein increased after thermal injury.
Liposomes decreased serum .alpha..sub.1-acid glycoprotein 1 and 5
days postburn (p<0.05; FIG. 7). There were no significant
differences in serum haptoglobin between liposome-treated animals
and controls. Type II acute phase protein increased after burn by
nearly 50%. There were no differences in serum
.alpha..sub.2-macroglobulin between liposome-treated animals and
control animals.
[0087] All measured cytokines increased after the thermal injury.
Serum IL-1.beta. decreased during the first day postburn and serum
TNF-.alpha. at 1 and 2 days postburn in rats receiving liposomes
compared to controls (p<0.05; FIGS. 8A, B). No change in serum
IL-6 was observed in liposome treated rats compared to
controls.
EXAMPLE 8
Biological Efficacy
[0088] All rats survived the 60% TBSA scald burn and the drug
injections with no deleterious side effects. Wound healing was
determined as follows; the wound eschar was left intact for the
first 28 days and then removed by gentle traction, with caution
taken not to disturb or destroy the healing edge along the
periphery. After removing the eschar, the animals were placed on a
standard surface and the wound area traced onto acetate sheets
along the well-demarcated re-epithelized and non-burned interface
and the leading edge of the neoepithelium. The areas of these
tracings were calculated by computerized planimetry (Sigma Scan and
Sigma Plot software). In addition, skin biopsies were taken from
the wound edge at 33 days after burn and light microscopic analysis
was performed using established techniques. Histological
measurements for linear skin re-epithelization used the HE staining
technique.
[0089] At 28 and 33 days after the thermal injury, rats receiving
the IGF-I cDNA construct showed a significant increase in
re-epithelization when compared with those receiving liposomes
alone or saline (p<0.05; FIG. 10). This enhancement in wound
healing is most likely due to a mitogenic stimulus of IGF-I to
keratinocytes and fibroblasts, as increased mitogenic activity was
found in rats treated with the IGF-I cDNA construct compared with
those receiving liposomes alone or saline. Rats receiving the IGF-I
cDNA construct had higher total serum protein levels and total
liver protein levels compared with rats treated with liposomes or
saline (p<0.05; Table 5).
5TABLE 5 Total protein concentration in serum and liver Saline
Liposomes IGF-I cDNA (n = 10) (n = 10) (n = 10) Serum (g/dl) 5.2
.+-. 0.1 5.3 .+-. 0.1 5.5 .+-. 0.1* Liver (mg/ml) 0.60 .+-. 0.01
0.61 .+-. 0.02 0.71 .+-. 0.03* Data presented as mean .+-. SEM.
*Significant difference vs. Liposomes and saline p < 0.05.
EXAMPLE 9
Procurement of Fetal Amnion Membrane Donor Tissue and Preparation
of Amnion and Chorion
[0090] After informed consent, history taking and screening for
potential risk factors such as cancer, infectious diseases, drug
abuse and sexual behavior, the presumptive donor is tested for
Hepatitis B and C, RPR, HIV1, HIV2 (as is routinely done by the SBI
skin bank and as defined by AATB). This testing is done at the time
of delivery and at a scheduled outpatient appointment 60-90 days
after delivery. Immediately after delivery, placentas with adherent
fetal membranes are obtained from suitable donors (according to the
guidelines of AATB). After preliminary washing with Ringer's
solution in the delivery room, the placenta is transferred to a
tissue culture medium (e.g., RPMI 1640) containing antibacterial
and antifungal agents and transported at 4.degree. C. to the
processing site.
[0091] The procurement steps are carried out using aseptic
technique such that bacterial and fungal contamination of the
product does not occur. Microbiological testing is performed with
each processing step so as to monitor contamination. Additional PCR
techniques can be performed on the tissue samples to also exclude
viral infection (e.g., HIV-1 or -2).
[0092] At the processing site (SBI tissue bank facility),
additional washing and flushing of the placenta with Ringer's
solution via the umbilical vessels is carried out. Amnion and
chorion layers are then mechanically separated from each other and
from the placenta, and the covering cellular material removed by
enzymatic digestion with trypsin (Boehringer Mannheim,
Indianapolis, Ind.) (1:1 dilution of distilled water and trypsin
for 2 h at 20.degree. C.) followed by repeated rinsing with
phosphate buffered saline (according to WO 93/10722). For
consistency, prepared amnion is stored using standard control rate
freezing techniques. To obtain different tissue properties and
characteristics, fetal membrane structures are cross-linked using a
1.5% glutaraldehyde solution (Sigma, St. Louis, Mo.) for 20 min at
20.degree. C., followed by washing in 1.5% glycine (Sigma, St.
Louis, Mo.) 3 times for 15 min as described for skin preservation
by the Euro Skin Bank (61).
[0093] Preparation for definitive storage includes lyophilization
using a shelf freeze drying system with a double freeze drying
procedure which results in a moisture content of less than or equal
to 6% water, or preservation by shaking the fetal membranes in 85%
glycerol at 20.degree. C. for two 3 hour periods. The latter allows
for a residual moisture of 15% water and assures inactivation of
HIV and other viruses (62). The latter treatment results in no
remaining viable cells and a diminished antigenicity resulting in
decreased immunologic response (63, 64). Glycerol-preserved tissue
is stored in glycerol at 4.degree. C. Both procedures are completed
by transfer of the tissue into a foil bag, allowing for further
sterilization by gamma radiation or ethylene gas. Before use, the
tissue is reconstituted by immersion in saline/Ringer's solution.
Additional wound coverage materials, such as a synthetic fabric
comprising type I and II collagens and human skin, are prepared for
comparison purposes (U.S. Pat. No. 5,002,071; Walther et al.,
1998).
EXAMPLE 10
Cholesterol-containing Cationic Liposomes as a Delivery System for
Gene Therapy in Trauma Attenuate the Acute Phase Response in
Thermally Injured Rats
[0094] One major contributor to the hypermetabolism associated with
a thermal injury is the increase in acute phase proteins and
cytokines [9, 10, 11]. It was shown herein that administration of
cholesterol-containing cationic liposomes improved body weight and
the expression of the constitutive hepatic proteins transferrin and
total protein after burn compared to saline. It was further shown
that liposomes decrease serum type I acute phase proteins and the
type I acute phase proteins responsive pro-inflammatory cytokines
serum TNF-.alpha. and IL-1.beta.. It is likely that the decrease in
IL-1.beta. and TNF-.alpha. subsequently leads to a decrease in
these acute phase proteins.
[0095] The reasons for the beneficial effects associated with
liposomes are most likely due to the direct effect of the liposomal
lipid moieties on damaged cell membranes or to an enhancement of
the uptake of extracellular nutrients and the in situ encapsulation
and protection of the endogenous growth factors and cytokines
elaborated locally as part of the hypermetabolic response which is
triggered by acute phase proteins and cytokines. Pro-inflammatory
cytokines, in particular TNF-.alpha., inhibit protein synthesis and
induce weight loss [16-18]. After a thermal injury, as well as in
sepsis, TNF-.alpha. serum levels increase along with sepsis-induced
muscle proteolysis [19-21]. Decreased serum TNF-.alpha. levels are
associated with an improvement in net protein balance and a
reduction in body weight loss in thermally injured pediatric
patients [22, 23]. Thus, as shown herein, a decrease in serum
TNF-.alpha. may preserve body weight and increase serum transferrin
levels.
[0096] Several studies have attempted to determine the mechanisms
by which cationic liposomes exert an inhibitory effect on cytokine
expression in vitro, however the exact mechanisms are currently not
defined [25-30]. It seems likely that nuclear factor kappa B
(NF-.kappa.B) plays an important role [27]. NF-.kappa.B is a
transcription signal and is crucial in the development of the
cellular immune and inflammatory response [27]. Due to
electrostatic interactions between cells and cationic liposomes,
the liposomes bind to the receptor for oxidized low-density
lipoproteins (OxLDL) [28, 29]. Sambrand and colleagues have shown
that this competitive binding and subsequent activation of the
OxLDL receptor indirectly suppresses activation and/or binding of
NF-.kappa.B to its cognate DNA site [28]. Furthermore, Aramaki and
colleagues have shown that cationic liposomes inhibit tyrosine
phosphorylation of p41, a protein of the MAP kinases transcription
factor family, which consecutively leads to a down-regulation of
NF-.kappa.B [25-27]. The inhibition of p41 and NF-.kappa.B inhibits
the induction of inducible nitric oxide synthetase (iNOS), which
decreases subsequent nitric oxide (NO) expression [25, 26]. These
changes in the signal pathway have been hypothesized to be
responsible for the selective inhibition of TNF-.alpha. expression
at the post-transcriptional level [25, 26, 31]. Given that NO and
iNOS stimulate IL-1.beta. expression, it seems likely that the
inhibition of NO or iNOS activity through cationic liposomes leads
to decreased IL-1.beta. expression [32, 33]. Therefore, cationic
liposome administration apparently proves beneficial in the
treatment of inflammatory diseases, in part because of their
inhibition of pro-inflammatory cytokine release [31, 34].
[0097] The administration of cholesterol-containing cationic
liposomes modulates the hypermetabolic response by affecting
cytokine expression, although the effects of the injection are not
likely to persist beyond 5 days. There were no differences in serum
cytokine and protein 7 days postburn between liposome-treated and
control animals. Furthermore, serum transferrin decreased from day
5 to day 7 postburn in rats receiving liposomes, whereas
control-treated animals showed an increase in serum transferrin
from day 5 to day 7 postburn. In addition, serum haptoglobin
increased from day 5 to 7 in the liposome-treated group, while
serum haptoglobin decreased over the same time period in control
animals. Therefore, cholesterol-cationic liposomes increased
constitutive hepatic proteins and decreased type I acute phase
proteins, with associated decreases in IL-1.beta. and TNF-.alpha.
levels. Thus, cholesterol-cationic liposomes appear to be suitable
as a delivery system for gene therapy in trauma, because liposomes
favorably modulate the trauma-induced hypermetabolic response and
do not display the cytotoxicity typically associated with the use
of other cationic liposomes in vivo [4, 7].
EXAMPLE 11
Clinical Impact of Multiple Injections of an IGF-I Gene Construct
After Thermal Injury
[0098] All rats in each group survived the 60% TBSA scald burn and
drug injections with no evidence of any deleterious side effects.
Total body weight increased at nearly 2% per week for the first 4
weeks postburn in animals transfected with single and multiple
injections of liposomes-IGF-I cDNA construct, with no differences
between the two groups. Rats receiving multiple injections of the
IGF-I cDNA construct had higher serum protein levels (single
injection 5.2.+-.0.05 g/dl vs. multiple injection 5.5.+-.0.06 g/dl)
and total liver protein (single injection 0.64.+-.0.002 mg/ml vs.
multiple injection 0.71.+-.0.03 mg/dl) compared to a single
injection (p<0.05). After the eschar was removed, the percent
area of burn wound re-epithelization was significantly larger 33
days after burn in rats receiving the double injection of IGF-I
cDNA compared to the single injection (38.+-.2% vs. 31.+-.2%
respectively; p<0.05). These results were confirmed by
histological measurements of linear re-epithelization. Rats treated
with double injections of IGF-I cDNA showed significantly more
re-epithelization when compared to single injections (p<0.05;
FIG. 11).
[0099] Transfection, determined by chemiluminescent reporter gene
assay to detect .beta.-galactosidase, was increased around the
wound perimeter in animals receiving multiple injections of
liposome encapsulated Lac Z cDNA and IGF-I cDNA constructs when
compared to single injections (p<0.05; FIGS. 12A, B). Skin
concentrations of IGF-I protein decreased from the skin biopsy
point I to the skin biopsy point III in rats receiving the single
injection of IGF-I cDNA construct along the wound edge (FIG. 13A).
Animals receiving double injections of the cDNA construct showed
consistent elevated IGF-I protein concentrations along the entire
wound edge (FIG. 13B). Additionally, transfection was detectable as
early as one day after subcutaneous injection of the LacZ gene. The
rate of transfection increased and peaked 5 days after injection.
In contrast to in vitro experiments in which transfection was not
detectable 7 days after administration, transfection of skin cells
was still detectable 7 and 14 days after in vivo injection.
[0100] After the subcutaneous injection of the IGF-I cDNA and the
reporter Lac Z construct, transfected dermal cells, myofibroblasts,
endothelial cells, and macrophages were identified, including
multinucleate giant cells known to be proliferative. mRNA levels
for IGF-I were increased in the skin of rats transfected with the
IGF-I construct. That the mRNA was translated into protein is
consistent with the observed increase in skin IGF-I protein
concentrations. This transient increase in the local expression of
IGF-I protein could cause a concurrent stimulation of IGFBP-3
protein synthesis and increased levels of the biologically active
complex IGF-I/IGFBP-3 locally without any concomitant
supraphysiological increases in circulating levels of free IGF-I
protein, and therefore, no deleterious side effects [49, 50]. The
small amounts of IGF-I protein expressed after liposomal
transfections are effective in a paracrine fashion without the
adverse effects of larger dosages required by systemic
administration.
[0101] An elevation of IGF-I protein concentration in the skin
improved wound healing in terms of re-epithelization and dermal
cell recovery along with dermal cell mitosis. Rats receiving the
IGF-I cDNA also had increased body weight and total protein
concentrations in serum and liver. As neither transfection nor
increased IGF-I expression was observed in blood, liver, spleen or
kidney, the beneficial effects (e.g., preserved body weight,
increased serum and liver protein concentration) are due to
enhanced wound healing and improved dermal cell recovery after
injury and result from the paracrine effects associated with higher
local levels of IGF-I protein as opposed to changes in circulating
levels of IGF-I protein resulting from systemic transfection. IGF-I
exerted mitogenic effects on keratinocytes and fibroblasts with the
stimulation of collagen synthesis, as well as improved cell
recovery after injury, leading to enhanced wound healing [40, 41,
48].
[0102] The advantages of early wound closure, demonstrated in
several clinical studies [36, 37], include a diminished
hypermetabolic burn response and a decrease in inflammatory
mediators, such as IL-1, IL-6, IL-8, and TNF.alpha. [36, 37].
Furthermore, IGF-I protein decreases pro-inflammatory cytokines
IL-1.beta. and TNF-.alpha. expression after thermal injury [57].
Therefore, IGF-I may exert its systemic beneficial effect through
the enhancement of re-epithelialization and/or the decrease of the
pro-inflammatory response in the skin, which is one of the major
sources of cytokine synthesis and release after burn [58, 59].
[0103] It is demonstrated herein that transfection is restricted to
the skin within a small perimeter of the sites of injection. The
nature of the expression of IGF-I was most likely due to
interactions between the positive surface charges on cationic
liposomes and negatively charged outer cell membranes, which
restricted liposomal migration [54]. However, transfection and
IGF-I expression was significantly increased when multiple
injection sites were used when compared to a single injection site.
Multiple injections of IGF-I cDNA demonstrated a consistent
elevation of IGF-I protein expression along with improved wound
healing, whereas a single injection demonstrated less IGF-I protein
along the wound. This finding is clinically relevant, because the
liposomes encapsulating the gene should be applied at well-defined
distances from trauma sites to provide optimal transfection and
protein expression.
[0104] Experiments herein demonstrate that the subcutaneous
administration of liposome-encapsulated IGF-I cDNA constructs
successfully transfected dermal cells. It is also demonstrated that
the cDNA was transcribed into mRNA and translated into IGF-I
protein. Multiple injections of the IGF-I cDNA increased the number
of transfected cells, and protein expression, which improved wound
healing when compared to a single injection. The process of
transfection, transcription and translation was restricted to the
skin, as systemic transfection or increased systemic IGF-I protein
expression was not observed. The biological responses to increased
skin IFG-I were an enhancement in wound healing with subsequent
systemic improvements to the hypermetabolic response.
EXAMPLE 12
Liposome-encapsulated IGF-I cDNA Gene Transfer Increases IGF-I
Protein in Skin Cells to Promote Wound Healing
[0105] The use of transient gene therapy after trauma is a new
approach to improve clinical outcome and mortality. Of major
importance is the selection of the appropriate vector for gene
delivery [1, 2]. Non-viral composition, non-cytotoxicity, increased
infectivity and anti-inflammatory activity make
cholesterol-containing cationic liposomes a promising approach to
ameliorate the burn-induced hypermetabolic response [4, 51-53]. It
has been shown herein that cholesterol-containing cationic
liposomes are an effective delivery system in vivo after thermal
injury. Additionally, the mechanisms by which DNA transfection and
subsequent induced gene expression alter thermal trauma responses
are reported herein, as well as the effects of IGF-I gene transfer
in a model of thermal injury. It was shown herein that the
subcutaneous administration of liposome-encapsulated IGF-I cDNA
constructs successfully transfected dermal cells and that the cDNA
was transcribed into mRNA and translated into IGF-I protein. The
process of transfection, transcription and translation was
restricted to the skin. The biological responses to increased skin
IGF-I were an enhancement in wound healing with subsequent systemic
improvements to the hypermetabolic response. From these findings,
it is concluded that cholesterol-containing cationic liposomes
encapsulating an expression plasmid vector for IGF-I cDNA, when
given to rats with a 60% TBSA thermal injury, are effective in
increasing IGF-I skin protein concentrations and thereby enhancing
wound healing.
EXAMPLE 13
Comparison of Membranes for Cover Material
[0106] To determine the biological efficacy of the different wound
coverage materials, outcome measurements are taken weekly over a
time period of 4 months and include: a) wound healing time; b)
re-epithelization by computerized planimetry, histological/electron
microscopic measurements; c) integrity by take rate and histology
and immune markers of rejection; and d) elastic rates and
contraction. Based on this data, the functionality and the efficacy
of the different cover materials are evaluated and the most
suitable determined.
[0107] Furthermore, gene transfer is measured by: a) histochemical
and chemiluminescent assays of reporter gene (.beta.-galactosidase)
expression as a screen for transfection; b) northern blot analysis
for IGF-I mRNA in the skin as a marker for transcription; and c)
radioimmunoassays to detect the IGF-I protein in the skin.
[0108] Additionally, the acute phase response is determined by: a)
body weight, nutritional intake, nitrogen balance and protein
concentration in muscle and liver; b) constitutive hepatic protein
production (albumin, transferrin, pre-albumin and retinol-binding
protein) determined by nephelometer; c) acute phase proteins
(haptoglobin, .alpha.1-acid-glycoprotein, .alpha.2-macroglobulin
and fibrinogen) determined by enzyme-linked immunoassays (ELISA)
and nephelometer; and e) cytokines (interleukin-1.beta.,
interleukin-4, interleukin-6, interleukin-8, interleukin-10,
tumor-necrosis factor-.alpha. and interferon-.gamma.) determined by
ELISA.
EXAMPLE 14
Wound Coverage of a Burn Injury
[0109] To more accurately simulate clinical treatment, each of the
15 Yorkshire swines receive a 35% total body surface area (TBSA)
full-thickness flame burn (standard model) under anesthesia and
analgesia. The animals remain restrained in a hammock for 24 hours
and receive intensive care, including adequate resuscitation and
analgesia. To mimic clinical setting, twenty-four hours after
receiving the burn, the animals undergo wound excision and
grafting. This is approximately the same time frame after thermal
injury that human patients are excised and grafted. Under general
anesthesia and analgesia, the entire burn wound is excised and
immediately covered with either human amnion or human amnion
impregnated with liposomal gene complexes. Only one type of cover
material is used per animal, as opposed to different types of wound
coverage material on the dorsum of the same animal. The cover is
stapled to the unburned wound and covered with gauze containing
triple-antibiotic ointment and a bulky pressure dressing. Staples
and/or sutures are used as required. Following surgery, the study
animals were suspended in a hammock restraint while recovering from
anesthesia as described above.
[0110] These experiments determine the effect of different cover
materials with and without the liposomal expression vectors on the
hepatic acute phase response and correlates those effects to wound
healing. Therefore, immediately after burn, surgery, and at
post-operative days 2, 4, 6, 8, 10, 12, 14 and 16, venous blood is
drawn from the animal and examined for constitutive hepatic
proteins, acute phase proteins and cytokines. The human amnion or
INTEGRA.TM. represents a reservoir for liposomal gene constructs.
Due to electrostatic interactions between the cell/wound surface,
liposomes will migrate out of the impregnated cover material and
transfect skin and wounded cells to thereby increase local IGF-I
concentration. The increase in IGF-I concentration subsequently
improves wound healing.
EXAMPLE 15
Additional Growth Factors in Concert with IGF-I
[0111] The combination of fetal membrane or human cadaver skin
impregnated with liposomal gene constructs accelerate and improve
the graft take, functionality, wound healing and the hypermetabolic
response after injury. The liposomal gene construct(s) may encode
the insulin-like growth factor-I (IGF-I), keratinocyte growth
factor (KGF), growth hormone (GH), fibroblast growth factor (FGF),
epidermal growth factor (EGF), platelet derived growth factor
(PDGF), transforming growth factor-.beta. (TGF-.beta.), or any
combination of the above-mentioned factors.
Conclusion
[0112] Liposomes containing a cDNA encoding IGF-I have been shown
to preserve body weight after a 60% TBSA wound, prevent muscle
protein wasting, improve wound healing and increase IGF-I protein
concentration in the skin. Furthermore, cell transfection and
subsequent IGF-I gene expression is a local event restricted to the
injection site. As no significant differences were detected in
serum IGF-I and IGFBP-3 concentrations, the beneficial effects of
the liposome gene delivery are due to localized enhanced wound
healing, rather than systemic changes in circulating IGF-I.
[0113] The present invention demonstrates that cDNAs encoding
growth-enhancing agents favorably modulates the hypermetabolic
response after thermal injury, and that liposome constructs can be
used effectively as a delivery system for gene transfections. The
therapeutic benefits of liposomal gene transfection was further
enhanced by increasing the number of injection sites around the
wound edges or by "wound dressing", which increased the number of
transfected cells and the concomitant levels of gene expression.
Therefore, fetal membrane, or other wound coverage materials,
impregnated with liposomes containing the IGF-I gene construct
improves wound healing and represents the optimal burn wound
treatment. This application of liposomal gene therapy is used to
impregnate human cadaver skin, amnion or other types of wound
coverage and/or closure materials and may revolutionize plastic,
reconstructive, trauma and burn surgery and improve the clinical
outcome of those patients.
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[0180] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Further, these patents and publications are
incorporated by reference herein to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0181] One skilled in the art will appreciate readily that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those objects,
ends and advantages inherent herein. The present examples, along
with the methods, procedures, treatments, molecules, and specific
compounds described herein are presently representative of
preferred embodiments, are exemplary, and are not intended as
limitations on the scope of the invention. Changes therein and
other uses will occur to those skilled in the art which are
encompassed within the spirit of the invention as defined by the
scope of the claims.
* * * * *