In vivo gene transfer methods for wound healing

The present invention relates to an in vivo method for specific targeting and transfer of DNA into mammalian repair cells. The transferred DNA may include any DNA encoding a therapeutic protein of interest. The invention is based on the discovery that mammalian repair cells proliferate and migrate into a wound site where they actively take up and express DNA. The invention further relates to pharmaceutical compositions that may be used in the practice of the invention to transfer the DNA of interest. Such compositions include any suitable matrix in combination with the DNA of interest.

TABLE OF CONTENTS 
1. INTRODUCTION 
2. BACKGROUND OF INVENTION 
2.1 WOUND HEALING 
2.2 GENE THERAPY 
3. SUMMARY OF THE INVENTION 
3.1 DEFINITIONS 
4. DESCRIPTION OF THE DRAWINGS 
5. DETAILED DESCRIPTION OF THE INVENTION 
5.1 THE GENE ACTIVATED MATRIX 
5.1.1 THE MATRIX MATERIALS 
5.1.2 THE DNA 
5.1.3 PREATION OF THE GENE ACTIVATED MATRICES 
5.2. USES OF THE GENE ACTIVATED MATRIX 
5.3. BONE REGENERATION 
5.4. SOFT TISSUES 
5.5. ORGAN REGENERATION 
5.6. REGULATION OF ANGIOGENESIS 
5.7. REPAIR OF THE SKIN 
6. EXAMPLE: IMPLANT MATERIAL FOR USE IN BONE GENE TRANSFER 
7. EXAMPLE: IN VIVO PROTEIN DETECTION FOLLOWING TRANSGENE EXPRESSION 
7.1. .beta.-GALACTOSIDASE TRANSGENE 
7.2. LUCIFERASE TRANSGENE 
7.3. PTH TRANSGENES 
7.4. BMP TRANSGENE 
8. EXAMPLE: TRANSFER OF AN OSTEOTROPIC GENE STIMULATES BONE 
REGENERATION/REPAIR IN VIVO 
9. EXAMPLE: DIRECT GENE TRANSFER INTO REGENERATING BONE IN VIVO 
9.1. MATERIALS AND METHODS 
9.1.1. MAMMALIAN HOST MODEL 
9.1.2. IMMUNOHISTOCHEMISTRY 
9.1.3. LUCIFERASE AND .beta.-gal ENZYME ASSAYS 
9.1.4. pGAM1 EXPRESSION PLASMID 
9.1.5. pGAM2 EXPRESSION PLASMID 
9.1.6. PREATION OF GENE ACTIVATED COLLAGEN SPONGES 
9.1.7. RADIOGRAPHY 
9.2. RESULTS 
9.2.1. OSTEOTOMY MODEL 
9.2.2. MARKER GENE STUDIES 
9.2.3. BMP-4 GENE TRANSFER 
9.2.4. TRANSFER AND EXPRESSION OF A PLASMID COCKTAIL (BMP-4+PTH1-34) 
10. EXAMPLE: TRANSFER OF GENES TO REGENERATING TENDON AND TO REGENERATING 
CRUCIATE LIGAMENT IN VIVO 
10.1. MATERIALS AND METHODS 
10.2. RESULTS 
11. EXAMPLE: ADENOVIRAL GENE TRANSFER INTO REGENERATING BONE IN VIVO 
11.1. MATERIALS AND METHODS 
11.2. RESULTS 
12. EXAMPLE: TRANSFER OF GENES TO SKELETAL MUSCLE 
12.1. MATERIALS AND METHODS 
12.1.1 PREATION OF DNA-PLGA COATING COMPOSITION 
12.1.2 COATING A SURGICAL SUTURE 
12.1.3 REPAIRING SKELETAL MUSCLE WITH THE COATED SUTURE 
12.2 RESULTS 
13. EXAMPLE: TRANSFER OF GENES TO BLOOD VESSEL 
13.1. MATERIALS AND METHODS 
13.2. RESULTS 
1. INTRODUCTION 
The present invention relates to a novel in vivo method for the 
presentation and direct transfer of DNA encoding a therapeutic protein of 
interest into mammalian repair cells. The method involves implanting a 
matrix containing DNA of interest (referred to herein as a "gene activated 
matrix") into a fresh wound site. Repair cells, which normally originate 
in viable tissue surrounding the wound, proliferate and migrate into the 
gene activated matrix, wherein they encounter, take up and express the 
DNA. Transfected repair cells, therefore act, as in situ bioreactors 
(localized within the wound site) which produce agents (DNA-encoded RNAs, 
proteins, etc.) that heal the wound. 
The invention further relates to pharmaceutical compositions that may be 
used in the practice of the invention to transfer the DNA of interest. 
Such compositions include any suitable matrix in combination with the DNA 
of interest. 
2. BACKGROUND OF INVENTION 
2.1 WOUND HEALING 
Currently available wound healing therapies involve the administration of 
therapeutic proteins. Such therapeutic proteins may include regulatory 
factors involved in the normal healing process such as systemic hormones, 
cytokines, growth factors and other proteins that regulate proliferation 
and differentiation of cells. Growth factors, cytokines and hormones 
reported to have such wound healing capacity include, for example, the 
transforming growth factor-.beta. superfamily (TGF-.beta.) of proteins 
(Cox, D. A., 1995, Cell Biology International, 19:357-371) acidic 
fibroblast growth factor (FGF) (Slavin, J., 1995, Cell Biology 
International, 19:431-444), macrophage-colony stimulating factor (M-CSF) 
and calcium regulatory agents such as parathyroid hormone (PTH). 
A number of problems are associated with the use of therapeutic proteins, 
i.e. cytokines, in wound healing therapies. First, the purification and/or 
recombinant production of therapeutic proteins is often an expensive and 
time-consuming process. Despite best efforts, however, purified protein 
preparations are often unstable making storage and use cumbersome, and 
protein instability can lead to unexpected inflammatory reactions (to 
protein breakdown products) that are toxic to the host. 
Second, systemic delivery of therapeutic proteins, i.e. cytokines, can be 
associated with serious unwanted side effects in unwounded tissue. Due to 
inefficient delivery to specific cells and tissues in the body, 
administration of high doses of protein are required to ensure that 
sufficient amounts of the protein reach the appropriate tissue target. 
Because of the short half life in the body due to proteolytic degradation, 
the proteins must also be administered repeatedly which may give rise to 
an immune reaction to the therapeutic proteins. The circulation of high 
doses of therapeutic proteins is often toxic due to pleiotropic effects of 
the administered protein, and may give rise to serious side effects. 
Third, exogenous delivery of recombinant proteins is inefficient. Attempts 
have been made to limit the administration of high levels of protein 
through immobilization of therapeutic protein at the target site. However, 
this therapeutic approach complicates the readministration of the protein 
for repeated dosing. 
Fourth, for a variety of proteins such as membrane receptors, transcription 
factors and intracellular binding proteins, biological activity is 
dependant on correct expression and localization in the cell. For many 
proteins, correct cellular localization occurs as the protein is 
post-translationally modified inside the cells. Therefore, such proteins 
cannot be administered exogenously in such a way as to be taken up and 
properly localized inside the cell. 
As these problems attest, current recombinant protein therapies for wound 
healing are flawed, because they do not present a rational method for 
delivery of exogenous proteins. These proteins, i.e. cytokines, are 
normally produced at their site of action in physiological amounts and 
efficiently delivered to cell surface signaling receptors. 
2.2 GENE THERAPY 
Gene therapy was originally conceived of as a specific gene replacement 
therapy for correction of heritable defects to deliver functionally active 
therapeutic genes into targeted cells. Initial efforts toward somatic gene 
therapy have relied on indirect means of introducing genes into tissues, 
called ex vivo gene therapy, e.g., target cells are removed from the body, 
transfected or infected with vectors carrying recombinant genes, and 
re-implanted into the body ("autologous cell transfer"). A variety of 
transfection techniques are currently available and used to transfer DNA 
in vitro into cells; including calcium phosphate-DNA precipitation, 
DEAE-Dextran transfection, electroporation, liposome mediated DNA transfer 
or transduction with recombinant viral vectors. Such ex vivo treatment 
protocols have been proposed to transfer DNA into a variety of different 
cell types including epithelial cells (U.S. Pat. No. 4,868,116; Morgan and 
Mulligan WO87/00201; Morgan et al., 1987, Science 237:1476-1479; Morgan 
and Mulligan, U.S. Pat. No. 4,980,286), endothelial cells (WO89/05345), 
hepatocytes (WO89/07136; Wolff et al., 1987, Proc. Natl. Acad. Sci. USA 
84:3344-3348; Ledley et al., 1987 Proc. Natl. Acad. Sci. 84:5335-5339; 
Wilson and Mulligan, WO89/07136; Wilson et al., 1990, Proc. Natl. Acad. 
Sci. 87:8437-8441) fibroblasts (Palmer et al., 1987, Proc. Natl. Acad. 
Sci. USA 84:1055-1059; Anson et al., 1987, Mol. Biol. Med. 4:11-20; 
Rosenberg et al., 1988, Science 242:1575-1578; Naughton & Naughton, U.S. 
Pat. No. 4,963,489), lymphocytes (Anderson et al., U.S. Pat. No. 
5,399,346; Blaese, R. M. et al., 1995, Science 270:475-480) and 
hematopoietic stem cells (Lim, B. et al. 1989, Proc. Natl. Acad. Sci. USA 
86:8892-8896; Anderson et al., U.S. Pat. No. 5,399,346). 
Direct in vivo gene transfer has recently been attempted with formulations 
of DNA trapped in liposomes (Ledley et al., 1987, J. Pediatrics 110:1); or 
in proteoliposomes that contain viral envelope receptor proteins (Nicolau 
et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1068); and DNA coupled to a 
polylysine-glycoprotein carrier complex. In addition, "gene guns" have 
been used for gene delivery into cells (Australian Patent No. 9068389). It 
has even been speculated that naked DNA, or DNA associated with liposomes, 
can be formulated in liquid carrier solutions for injection into 
interstitial spaces for transfer of DNA into cells (Felgner, WO90/11092). 
Perhaps one of the greatest problems associated with currently devised gene 
therapies, whether ex vivo or in vivo, is the inability to transfer DNA 
efficiently into a targeted cell population and to achieve high level 
expression of the gene product in vivo. Viral vectors are regarded as the 
most efficient system, and recombinant replication-defective viral vectors 
have been used to transduce (i.e., infect) cells both ex vivo and in vivo. 
Such vectors have included retroviral, adenovirus and adeno-associated and 
herpes viral vectors. While highly efficient at gene transfer, the major 
disadvantages associated with the use of viral vectors include the 
inability of many viral vectors to infect non-dividing cells; problems 
associated with insertional mutagenesis; inflammatory reactions to the 
virus and potential helper virus production, and/or production and 
transmission of harmful virus to other human patients. 
In addition to the low efficiency of most cell types to take up and express 
foreign DNA, many targeted cell populations are found in such low numbers 
in the body that the efficiency of presentation of DNA to the specific 
targeted cell types is even further diminished. At present, no protocol or 
method, currently exists to increase the efficiency with which DNA is 
targeted to the targeted cell population. 
3. SUMMARY OF THE INVENTION 
The present invention relates to a novel method for specific targeting and 
transfer of DNA into mammalian repair cells involved in wound healing in 
order to express therapeutic products at the wound site. The method of the 
invention involves administering a gene activated matrix into a fresh 
wound site in the body. In this setting, repair cells are localized to the 
wound site, where they become transfected and eventually produce 
DNA-encoded agents (RNAs, proteins, etc.) that enhance wound healing. 
The invention is based, in part, on the discovery that repair cells, active 
in the wound healing process, proliferate and migrate from surrounding 
tissue into the area of the wound and infiltrate the gene activated 
matrix. The matrix acts as a scaffolding that promotes cell ingrowth, and, 
in turn, gene transfer, through the local accumulation of repair cells 
near the DNA. While in the matrix, repair cells are surprisingly efficient 
at taking up the DNA and expressing it as translational products, i.e., 
proteins, or transcriptional products, i.e., antisense and ribozymes. The 
transfected repair cells then serve as local bioreactors amplifying the 
production of the gene product in vivo. 
While any number of DNA sequences can be used in the method, preferred DNA 
sequences are those that encode translational products (i.e. proteins) or 
transcriptional products (i.e. antisense or ribozymes) that (a) promote 
tissue repair; or (b) are capable of disrupting a disease process (thereby 
allowing normal tissue healing to take place). 
The invention overcomes the shortcomings of procedures currently used for 
wound healing involving the administration of therapeutic proteins. First, 
DNA, which is both stable and non-toxic, can be safely administered in 
high doses in vivo. Second, repeated administration, while possible, is 
not required. The cells which take up and express the DNA provide a supply 
of gene product at the site of the wound. Third, the invention could be 
practiced in a way that addresses the temporal requirements of dosing. For 
example, the DNA can be presented in vectors that integrate into the 
genome of the targeted cell. In this case, all daughter cells will contain 
and express the transferred DNA thereby acting as a continuous source for 
the therapeutic agent. In contrast, non-integrating systems may be 
utilized wherein the DNA does not integrate into the genome and the gene 
is not passed on to daughter cells. In such an instance, when the wound 
healing process is completed and the gene product is no longer needed, the 
gene product will not be expressed. 
The invention is demonstrated by way of examples, which show that genes can 
be reproducibly transferred and expressed in a variety of wounded soft and 
hard tissues in vivo. Specifically, it is shown that the method of the 
invention overcomes the problems associated with currently available gene 
therapy protocols. The method of the invention provides gene transfer to a 
suitable number of repair cells to achieve functional effects, i.e., in 
the absence of any further targeting or cellular identification by the 
practitioner. In vivo methods of gene therapy require some form of 
targeting which very often does not work. In the method of the invention, 
targeting is not a problem. By analogy, the DNA acts much like "bait" in a 
"trap": the DNA is encountered by unwitting repair cells that have 
proliferated and then migrated into the gene activated matrix. These 
cells, in turn, are surprisingly capable of taking up DNA and expressing 
it as a therapeutic agent. 
In one embodiment of the invention, the method of the invention may be used 
as a drug delivery system through transfer of DNA into mammalian repair 
cells for the purpose of stimulating soft and hard tissue repair and 
tissue regeneration. The repair cells will be those cells that normally 
arrive at the area of the wound to be treated. Accordingly, there is no 
difficulty associated with the obtaining of suitable target cells to which 
the present therapeutic compositions should be applied. All that is 
required is the implantation of a gene activated matrix at the wound site. 
The nature of this biological environment is such that the appropriate 
repair cells will actively take up and express the "bait" DNA in the 
absence of any further targeting or cellular identification by the 
practitioner. 
In another embodiment, the method of the invention, using both biological 
and synthetic matrices, may be used to transfer DNA into mammalian repair 
cells to stimulate skeletal regeneration. In a further embodiment, the 
method of the invention, using both biological and synthetic matrices, may 
be used to transfer DNA into mammalian cells to stimulate ligament and 
tendon repair. The method of the invention may further be employed, using 
both biological and synthetic matrices to transfer DNA into mammalian 
repair cells to stimulate skeletal muscle repair and/or blood vessel 
repair. 
The DNA to be used in the practice of the invention may include any DNA 
encoding translational products (i.e. proteins) or transcriptional 
products (i.e. antisense or ribozymes) that promote tissue repair or are 
capable of disrupting a disease process. For example, the DNA may comprise 
genes encoding therapeutically useful proteins such as growth factors, 
cytokines, hormones, etc. Additionally, the DNA may encode anti-sense or 
ribozyme molecules that may inhibit the translation of mRNAs encoding 
proteins that inhibit wound healing or which induce inflammation. 
The DNA encoding the therapeutic product of interest is associated with, or 
impregnated within, a matrix to form a gene activated matrix. Once 
prepared, the gene activated matrix is placed within the mammal at the 
site of a wound. 
The invention is demonstrated by way of examples, wherein the efficient in 
vivo transfer and expression of genes into tissue undergoing repair and 
regeneration is demonstrated. 
3.1 DEFINITIONS 
As used herein, the following terms will have the meanings indicated below. 
A gene activated matrix (GAM) is defined herein as any matrix material 
containing DNA encoding a therapeutic agent of interest. For example, gene 
activated matrices are placed within wound sites in the body of a 
mammalian host to enhance wound healing. 
A repair cell is defined herein as any cell which is stimulated to migrate 
and proliferate in response to tissue injury. Repair cells are a component 
of the wound healing response. Such cells include fibroblasts, capillary 
endothelial cells, capillary pericytes, mononuclear inflammatory cells, 
segmented inflammatory cells and granulation tissue cells. 
A wound site is defined as any location in the host that arises from 
traumatic tissue injury, or alternatively, from tissue damage either 
induced by, or resulting from, surgical procedures.

5. DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to an in vivo method for presentation and 
transfer of DNA into mammalian repair cells for the purpose of expressing 
therapeutic agents. The method of the invention involves implanting or 
placing gene activated matrices into a fresh wound site. 
Wound healing is usually a coordinated, stereotyped sequence of events that 
includes (a) tissue disruption and loss of normal tissue architecture; (b) 
cell necrosis and hemorrhage; hemostasis (clot formation); (c) 
infiltration of segmented and mononuclear inflammatory cells, with 
vascular congestion and tissue edema; (d) dissolution of the clot as well 
as damaged cells and tissues by mononuclear cells (macrophages) (e) 
formation of granulation tissue (fibroplasia and angiogenesis). This 
sequence of cellular events has been observed in wounds from all tissues 
and organs generated in a large number of mammalian species (Gailet et 
al., 1994, Curr. Opin. Cell. Biol. 6:717-725). Therefore, the cellular 
sequence described above is a universal aspect of the repair of all 
mammalian tissues. 
The invention is based on the discovery that repair cells involved in the 
wound healing process will naturally proliferate and migrate to the site 
of tissue injury and infiltrate the gene activated matrix. Surprisingly, 
these repair cells, which are normally difficult to efficiently transfect, 
either in vitro or in vivo, are extremely efficient at taking up and 
expressing DNA when activated to proliferate by the wound healing process. 
Taking advantage of this feature, the methods of the present invention are 
designed to efficiently transfer, one or more DNA molecules encoding 
therapeutic agents to the proliferating repair cells. The methods involve 
the administration of a gene activated matrix containing DNA encoding 
translational products (i.e. therapeutic proteins) or transcriptional 
products (i.e. antisense or ribozymes) within a mammalian host at the site 
of a wound. The wound may arise from traumatic tissue injury, or 
alternatively, from tissue damage either induced by, or resulting from, 
surgical procedures. 
As the proliferating repair cells migrate into and contact a gene activated 
matrix, they take up and express the DNA of interest thereby amplifying 
the amount of the therapeutic agent, protein or RNA. The transfected 
repair cells thereby serve as local bioreactors producing therapeutic 
agents that influence the local repair environment. For example, growth 
factors or cytokines produced by the transfected repair cells, will bind 
and stimulate targeted effector cells that express cognate cell surface 
receptors, thereby stimulating and amplifying the cascade of physiological 
events normally associated with the wound healing process. 
Alternatively, the repair cells may take up and express DNA encoding 
proteins that inhibit the activity of antagonists of the wound healing 
process. The DNA may also encode antisense or ribozyme RNA molecules that 
may be used to inhibit translation of mRNAs encoding inflammatory proteins 
or other factors that inhibit wound healing or cause excessive fibrosis. 
The gene activated matrix of the invention can be transferred to the 
patient using a variety of techniques. For example, when stimulating wound 
healing and regeneration, the matrices are transferred directly to the 
site of the wound, i.e., the fractured bone, injured connective tissue, 
etc. For use in skin repair, the matrices will be topically administered. 
For use in organ regeneration, the matrices will be surgically placed in a 
wound made in the organ. 
Since the method of the invention is based on the natural migration and 
proliferation of repair cells into a wound site, and infiltration into the 
gene activated matrix located at the wound site, followed by the uptake of 
DNA, it is understood that the matrices must be transferred into a site in 
the body where the wound healing process has been induced. 
One particularly important feature of the present invention is that the 
repair process may be engineered to result in either the formation of scar 
tissue and/or tissue regeneration. For example, the overexpression of the 
therapeutic proteins at the site of the wound, may result in regeneration 
of the injured tissue without the formation of scar tissue. In many 
instances, for example, such as bone repair, such regeneration is 
desirable because scar tissue is not optimally designed to support normal 
mechanical function. Alternatively, around a suture it may be desirable to 
form scar tissue to hold inherently weak tissue together. Therefore, the 
methods of invention may be used to stimulate wound healing either with, 
or without, the formation of scar tissue depending on the type and level 
of therapeutic protein expressed. 
Direct plasmid DNA transfer from a matrix to a mammalian repair cell, 
through stimulation of the wound healing process, offers a number of 
advantages. First, the ease of producing and purifying DNA constructs 
compares favorably with traditional protein production method cost. 
Second, matrices can act as structural scaffolds that, in and of 
themselves, promote cell ingrowth and proliferation. Thus, they facilitate 
the targeting of repair cells for gene transfer. Third, direct gene 
transfer may be an advantageous method of drug delivery for molecules that 
normally undergo complex biosynthetic processing or for receptors which 
must be properly positioned in the cellular membrane. These types of 
molecules would fail to work if exogenously delivered to cells. 
The present invention also relates to pharmaceutical compositions 
comprising matrices containing DNA for use in wound healing. The 
compositions of the invention are generally comprised of a biocompatible, 
or bone compatible, matrix material containing DNA encoding a therapeutic 
protein of interest. 
The invention overcomes shortcomings specifically associated with current 
recombinant protein therapies for wound healing applications. First, 
direct gene transfer is a rational strategy that allows transfected cells 
to (a) make physiological amounts of therapeutic protein, modified in a 
tissue- and context-specific manner, and (b) deliver this protein to the 
appropriate cell surface signaling receptor under the appropriate 
circumstances. For reasons described above, exogenous delivery of such 
molecules is expected to be associated with significant dosing and 
delivery problems. Second, repeated administration, while possible, is not 
required with gene activated matrix technology: cell uptake of DNA can be 
controlled precisely with well-established sustained release delivery 
technologies, or, alternatively, integration of transfected DNA can be 
associated with long term recombinant protein expression. 
The method of the invention can be universally applied to wounds that 
involve many different cells, tissues and organs; the repair cells of 
granulation tissue (Gailet et al., 1994, Curr. Opin. Cell. Biol. 
6:717-725) are "targeted" where the method of the invention is used. The 
invention is demonstrated herein in three animal models (dog, rat and 
rabbit) and five tissues (bone, tendon, ligament, blood vessel and 
skeletal muscle), using three marker genes (.beta.-galactosidase, 
luciferase and alkaline phosphatase), three promoter systems (CMV, RSV, 
LTR and SV40), two types of matrices (biological and synthetic). In all 
instances, repair cells that migrated into the gene activated matrix were 
successfully transfected. In particular, a functional outcome (bone 
growth) has been demonstrated following gene transfer to repair 
fibroblasts of a plasmid construct encoding either BMP-4, which acts as a 
signal transducing switch for osteoblast differentiation and growth 
(Wozney, 1992, Mol. Reprod. Dev. 32:160-167; Reddi, 1994, Curr. Opin. 
Genet. Deve. 4:737-744) or PTH1-34, which recruits osteoprogenitor cells 
(Orloff, et al, 1992, Endocrinology 131:1603-1611; Dempster et al., 1995 
Endocrin Rev. 4:247-250). 
5.1 THE GENE ACTIVATED MATRIX 
Any biocompatible matrix material containing DNA encoding a therapeutic 
agent of interest, such as a translational product, i.e. therapeutic 
proteins, or transcriptional products, i.e. antisense or ribozymes, can be 
formulated and used in accordance with the invention. 
The gene activated matrices of the invention may be derived from any 
biocompatible material. Such materials may include, but are not limited 
to, biodegradable or non-biodegradable materials formulated into scaffolds 
that support cell attachment and growth, powders or gels. Matrices may be 
derived from synthetic polymers or naturally occurring proteins such as 
collagen, other extracellular matrix proteins, or other structural 
macromolecules. 
The DNA incorporated into the matrix may encode any of a variety of 
therapeutic proteins depending on the envisioned therapeutic use. Such 
proteins may include growth factors, cytokines, hormones or any other 
proteins capable of regulating the growth, differentiation or 
physiological function of cells. The DNA may also encode antisense or 
ribozyme molecules which inhibit the translation of proteins that inhibit 
wound repair and/or induce inflammation. 
The transferred DNA need not be integrated into the genome of the target 
cell; indeed, the use of non-integrating DNA in the gene activated matrix 
is the preferred embodiment of the present invention. In this way, when 
the wound healing process is completed and the gene product is no longer 
needed, the gene product will not be expressed. 
Therapeutic kits containing a biocompatible matrix and DNA form another 
aspect of the invention. In some instances the kits will contain preformed 
gene activated matrices thereby allowing the physician, to directly 
administer the matrix within the body. Alternatively, the kits may contain 
the components necessary for formation of a gene activated matrix. In such 
cases the physician may combine the components to form the gene activated 
matrices which may then be used therapeutically by placement within the 
body. In an embodiment of the invention the matrices may be used to coat 
surgical devices such as suture materials or implants. In yet another 
embodiment of the invention, gene activated matrices may include ready to 
use sponges, tubes, band-aids, lyophilized components, gels, patches or 
powders and telfa pads. 
5.1.1 THE MATRIX MATERIALS 
In one aspect of the invention, compositions are prepared in which the DNA 
encoding the therapeutic agent of interest is associated with or 
impregnated within a matrix to form a gene activated matrix. The matrix 
compositions function (i) to facilitate ingrowth of repair cells 
(targeting); and (ii) to harbor DNA (delivery). Once the gene activated 
matrix is prepared it is stored for future use or placed immediately at 
the site of the wound. 
The type of matrix that may be used in the compositions, devices and 
methods of the invention is virtually limitless and may include both 
biological and synthetic matrices. The matrix will have all the features 
commonly associated with being "biocompatible", in that it is in a form 
that does not produce an adverse, allergic or other untoward reaction when 
administered to a mammalian host. Such matrices may be formed from both 
natural or synthetic materials. The matrices may be non-biodegradable in 
instances where it is desirable to leave permanent structures in the body; 
or biodegradable where the expression of the therapeutic protein is 
required only for a short duration of time. The matrices may take the form 
of sponges, implants, tubes, telfa pads, band-aids, bandages, pads, 
lyophilized components, gels, patches, powders or nanoparticles. In 
addition, matrices can be designed to allow for sustained release of the 
DNA over prolonged periods of time. 
The choice of matrix material will differ according to the particular 
circumstances and the site of the wound that is to be treated. Matrices 
such as those described in U.S. Pat. No. 5,270,300, incorporated herein by 
reference, may be employed. Physical and chemical characteristics, such 
as, e.g., biocompatibility, biodegradability, strength, rigidity, 
interface properties and even cosmetic appearance may be considered in 
choosing a matrix, as is well known to those of skill in the art. 
Appropriate matrices will both deliver the DNA molecule and also act as an 
in situ scaffolding through which mammalian repair cells may migrate. 
Where the matrices are to be maintained for extended periods of time, 
non-biodegradable matrices may be employed, such as sintered 
hydroxyapatite, bioglass, aluminates, other bioceramic materials and metal 
materials, particularly titanium. A suitable ceramic delivery system is 
that described in U.S. Pat. No. 4,596,574, incorporated herein by 
reference. The bioceramics may be altered in composition, such as in 
calcium-aluminate-phosphate; and they may be processed to modify 
particular physical and chemical characteristics, such as pore size, 
particle size, particle shape, and biodegradability. Polymeric matrices 
may also be employed, including acrylic ester polymers and lactic acid 
polymers, as disclosed in U.S. Pat. Nos. 4,521,909, and 4,563,489, 
respectively, each incorporated herein by reference. Particular examples 
of useful polymers are those of orthoesters, anhydrides, 
propylene-cofumarates, or a polymer of one or more .gamma.-hydroxy 
carboxylic acid monomers, e.g., .gamma.-hydroxy auric acid (glycolic acid) 
and/or .gamma.-hydroxy propionic acid (lactic acid). 
A particularly important aspect of the present invention is its use in 
connection with orthopaedic implants and interfaces and artificial joints, 
including implants themselves and functional parts of an implant, such as, 
e.g., surgical screws, pins, and the like. In preferred embodiments, it is 
contemplated that the metal surface or surfaces of an implant or a portion 
thereof, such as a titanium surface, will be coated with a material that 
has an affinity for nucleic acids, most preferably, with hydroxyl apatite, 
and then the coated-metal will be further coated with the gene or nucleic 
acid that one wishes to transfer. The available chemical groups of the 
absorptive material, such as hydroxyl apatite, may be readily manipulated 
to control its affinity for nucleic acids, as is known to those of skill 
in the art. 
In preferred embodiments, it is contemplated that a biodegradable matrix 
will likely be most useful. A biodegradable matrix is generally defined as 
one that is capable of being reabsorbed into the body. Potential 
biodegradable matrices for use in connection with the compositions, 
devices and methods of this invention include, for example, biodegradable 
and chemically defined calcium sulfate, tricalciumphosphate, 
hydroxyapatite, polyactic acid, polyanhidrides, matrices of purified 
proteins, and semipurified extracellular matrix compositions. 
Other biocompatible biodegradable polymers that may be used are well known 
in the art and include, by way of example and not limitation, polyesters 
such as polyglycolides, polylactides and polylactic polyglycolic acid 
copolymers ("PLGA")(Langer and Folkman, 1976, Nature 263:797-800); 
polyethers such as polycaprolactone ("PCL"); polyanhydrides; polyalkyl 
cyanoacrylates such as n-butyl cyanoacrylate and isopropyl cyanoacrylate; 
polyacrylamides; poly(orthoesters); polyphosphazenes; polypeptides; 
polyurethanes; and mixtures of such polymers. 
It is to be understood that virtually any polymer that is now known or that 
will be later developed suitable for the sustained or controlled release 
of nucleic acids may be employed in the present invention. 
In preferred embodiments, the biocompatible biodegradable polymer is a 
copolymer of glycolic acid and lactic acid ("PLGA") having a proportion 
between the lactic acid/glycolic acid units ranging from about 100/0 to 
about 25/75. The average molecular weight ("MW") of the polymer will 
typically range from about 6,000 to 700,000 and preferably from about 
30,000 to 120,000, as determined by gel-permeation chromatography using 
commercially available polystyrene of standard molecular weight, and have 
an intrinsic viscosity ranging from 0.5 to 10.5. 
The length of the period of continuous sustained or controlled release of 
nucleic acids from the matrix according to the invention will depend in 
large part on the MW of the polymer and the composition ratio of lactic 
acid/glycolic acid. Generally, a higher ratio of lactic acid/glycolic 
acid, such as for example 75/25, will provide for a longer period of 
controlled of sustained release of the nucleic acids, whereas a lower 
ratio of lactic acid/glycolic acid will provide for more rapid release of 
the nucleic acids. Preferably, the lactic acid/glycolic acid ratio is 
50/50. 
The length of period of sustained or controlled release is also dependent 
on the MW of the polymer. Generally, a higher MW polymer will provide for 
a longer period of controlled or sustained release. In the case of 
preparing, for example, matrices providing controlled or sustained release 
for about three months, when the composition ratio of lactic acid/glycolic 
acid is 100/0, the preferable average MW of polymer ranges from about 
7,000 to 25,000; when 90/10, from about 6,000 to 30,000; and when 80/20, 
from about 12,000 to 30,000. 
Another type of biomaterial that may be used is small intestinal submucosa 
(SIS). The SIS graft material may be prepared from a segment of jejunum of 
adult pigs. Isolation of tissue samples may be carried out using routine 
tissue culture techniques such as those described in Badybak et al., 1989, 
J. Surg. Res. 47:74-80. SIS material is prepared by removal of mesenteric 
tissue, inversion of the segment, followed by removal of the mucosa and 
superficial submucosa by a mechanical abrasion technique. After returning 
the segment to its original orientation, the serosa and muscle layers are 
rinsed and stored for further use. 
Another particular example of a suitable material is fibrous collagen, 
which may be lyophilized following extraction and partial purification 
from tissue and then sterilized. Matrices may also be prepared from tendon 
or dermal collagen, as may be obtained from a variety of commercial 
sources, such as, e.g., Sigma and Collagen Corporation. Collagen matrices 
may also be prepared as described in U.S. Pat. Nos. 4,394,370 and 
4,975,527, each incorporated herein by reference. 
In addition, lattices made of collagen and glycosaminoglycan (GAG) such as 
that described in Yannas & Burke, U.S. Pat. No. 4,505,266, may be used in 
the practice of the invention. The collagen/GAG matrix may effectively 
serve as a support or "scaffolding" structure into which repair cells may 
migrate. Collagen matrix, such as those disclosed in Bell, U.S. Pat. No. 
4,485,097, may also be used as a matrix material. 
The various collagenous materials may also be in the form of mineralized 
collagen. For example, the fibrous collagen implant material termed 
UltraFiber.TM., as may be obtained from Norian Corp., (1025 Terra Bella 
Ave., Mountain View, Calif., 94043) may be used for formation of matrices. 
U.S. Pat. No. 5,231,169, incorporated herein by reference, describes the 
preparation of mineralized collagen through the formation of calcium 
phosphate mineral under mild agitation in situ in the presence of 
dispersed collagen fibrils. Such a formulation may be employed in the 
context of delivering a nucleic acid segment to a bone tissue site. 
Mineralized collagen may be employed, for example, as part of gene 
activated matrix therapeutic kit for fracture repair. 
At least 20 different forms of collagen have been identified and each of 
these collagens may be used in the practice of the invention. For example, 
collagen may be purified from hyaline cartilage, as isolated from 
diarthrodial joints or growth plates. Type II collagen purified from 
hyaline cartilage is commercially available and may be purchased from, 
e.g., Sigma Chemical Company, St. Louis. Type I collagen from rat tail 
tendon may be purchased from, e.g., Collagen Corporation. Any form of 
recombinant collagen may also be employed, as may be obtained from a 
collagen-expressing recombinant host cell, including bacterial yeast, 
mammalian, and insect cells. When using collagen as a matrix material it 
may be advantageous to remove what is referred to as the "telopeptide" 
which is located at the end of the collagen molecule and known to induce 
an inflammatory response. 
The collagen used in the invention may, if desired be supplemented with 
additional minerals, such as calcium, e.g., in the form of calcium 
phosphate. Both native and recombinant type collagen may be supplemented 
by admixing, absorbing, or otherwise associating with, additional minerals 
in this manner. 
5.1.2 THE DNA 
The present methods and compositions may employ a variety of different 
types of DNA molecules. The DNA molecules may include genomic, cDNAs, 
single stranded DNA, double stranded DNA, triple stranded DNA, 
oligonucleotides and Z-DNA. 
The DNA molecules may code for a variety of factors that promote wound 
healing including extracellular, cell surface, and intracellular RNAs and 
proteins. Examples of extracellular proteins include growth factors, 
cytokines therapeutic proteins, hormones and peptide fragments of 
hormones, inhibitors of cytokines, peptide growth and differentiation 
factors, interleukins, chemokines, interferons, colony stimulating factors 
and angiogenic factors. Examples of such proteins include, but are not 
limited to, the superfamily of TGF-.beta. molecules, including the five 
TGF-.beta. isoforms and bone morphogenetic proteins (BMP), latent 
TGF-.beta. binding proteins, LTBP; keratinocyte growth factor (KGF); 
hepatocyte growth factor (HGF); platelet derived growth factor (PDGF); 
insulin-like growth factor (IGF); the basic fibroblast growth factors 
(FGF-1, FGF-2 etc.), vascular endothelial growth factor (VEGF); Factor 
VIII and Factor IX; erythropoietin (EPO); tissue plasminogen activator 
(TPA); activins and inhibins. Hormones which may be used in the practice 
of the invention include growth hormone (GH) and parathyroid hormone 
(PTH). Examples of extracellular proteins also include the extracellular 
matrix proteins such as collagen, laminin, and fibronectin. Examples of 
cell surface proteins include the family of cell adhesion molecules (e.g., 
the integrins, selectin, Ig family members such as N-CAM and L1, and 
cadherins); cytokine signaling receptors such as the type I and type II 
TGF-.beta. receptors and the FGF receptor; and non-signaling co-receptors 
such as betaglycan and syndecan. Examples of intracellular RNAs and 
proteins include the family of signal transducing kinases, cytoskeletal 
proteins such as talin and vinculin, cytokine binding proteins such as the 
family of latent TGF-.beta. binding proteins, and nuclear trans acting 
proteins such as transcription factors and enhancing factors. 
The DNA molecules may also code for proteins that block pathological 
processes, thereby allowing the natural wound healing process to occur 
unimpeded. Examples of blocking factors include ribozymes that destroy RNA 
function and DNAs that, for example, code for tissue inhibitors of enzymes 
that destroy tissue integrity, e.g., inhibitors of metalloproteinases 
associated with arthritis. 
One may obtain the DNA segment encoding the protein of interest using a 
variety of molecular biological techniques, generally known to those 
skilled in the art. For example, cDNA or genomic libraries may be screened 
using primers or probes with sequences based on the known nucleotide 
sequences. Polymerase chain reaction (PCR) may also be used to generate 
the DNA fragment encoding the protein of interest. Alternatively, the DNA 
fragment may be obtained from a commercial source. 
Genes with sequences that vary from those described in the literature are 
also encompassed by the invention, so long as the altered or modified gene 
still encodes a protein that functions to stimulate wound healing in any 
direct or indirect manner. These sequences include those caused by point 
mutations, those due to the degeneracies of the genetic code or naturally 
occurring allelic variants, and further modifications that have been 
introduced by genetic engineering, i.e., by the hand of man. 
Techniques for introducing changes in nucleotide sequences that are 
designed to alter the functional properties of the encoded proteins or 
polypeptides are well known in the art. Such modifications include the 
deletion, insertion or substitution of bases which result in changes in 
the amino acid sequence. Changes may be made to increase the activity of 
an encoded protein, to increase its biological stability or half-life, to 
change its glycosylation pattern, confer temperature sensitivity or to 
alter the expression pattern of the protein and the like. All such 
modifications to the nucleotide sequences are encompassed by this 
invention. 
The DNA encoding the translational or transcriptional products of interest 
may be recombinantly engineered into variety of vector systems that 
provide for replication of the DNA in large scale for the preparation of 
gene activated matrices. These vectors can be designed to contain the 
necessary elements for directing the transcription and/or translation of 
the DNA sequence taken up by the repair cells at the wound in vivo. 
Vectors that may be used include, but are not limited to those derived from 
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. For example, 
plasmid vectors such as pBR322, pUC 19/18, pUC 118, 119 and the M13 mp 
series of vectors may be used. Bacteriophage vectors may include 
.lambda.gt10, .lambda.gt11, .lambda.gt18-23, .lambda.ZAP/R and the EMBL 
series of bacteriophage vectors. Cosmid vectors that may be utilized 
include, but are not limited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, 
pMCS, pNNL, pHSG274, COS202, COS203, pWE15, pWE16 and the charomid 9 
series of vectors. Vectors that allow for the in vitro transcription of 
RNA, such as SP6 vectors, may also be used to produce large quantities of 
RNA that may be incorporated into matrices. Alternatively, recombinant 
virus vectors including, but not limited to those derived from viruses 
such as herpes virus, retroviruses, vaccinia viruses, adenoviruses, 
adeno-associated viruses or bovine papilloma virus may be engineered. 
While integrating vectors may be used, non-integrating systems, which do 
not transmit the gene product to daughter cells for many generations are 
preferred for wound healing. In this way, the gene product is expressed 
during the wound healing process, and as the gene is diluted out in 
progeny generations, the amount of expressed gene product is diminished. 
Methods which are well known to those skilled in the art can be used to 
construct expression vectors containing the protein coding sequence 
operatively associated with appropriate transcriptional/translational 
control signals. These methods include in vitro recombinant DNA 
techniques, and synthetic techniques. See, for example, the techniques 
described in Sambrook, et al., 1992, Molecular Cloning, A Laboratory 
Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al., 1989, 
Current Protocols in Molecular Biology, Greene Publishing Associates & 
Wiley Interscience, N.Y. 
The genes encoding the proteins of interest may be operatively associated 
with a variety of different promoter/enhancer elements. The expression 
elements of these vectors may vary in their strength and specificities. 
Depending on the host/vector system utilized, any one of a number of 
suitable transcription and translation elements may be used. The promoter 
may be in the form of the promoter which is naturally associated with the 
gene of interest. Alternatively, the DNA may be positioned under the 
control of a recombinant or heterologous promoter, i.e., a promoter that 
is not normally associated with that gene. For example, tissue specific 
promoter/enhancer elements may be used to regulate the expression of the 
transferred DNA in specific cell types. Examples of transcriptional 
control regions that exhibit tissue specificity which have been described 
and could be used, include but are not limited to: elastase I gene control 
region which is active in pancreatic acinar cells (Swift et al., 1984, 
Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. 
Biol. 50:399-409; MacDonald, 1987, Hepatology 7:42S-51S); insulin gene 
control region which is active in pancreatic beta cells (Hanahan, 1985, 
Nature 315:115-122); immunoglobulin gene control region which is active in 
lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adams et al., 
1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 
7:1436-1444): albumin gene control region which is active in liver 
(Pinkert et al., 1987, Genes and Devel. 1:268-276) alpha-fetoprotein gene 
control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. 
Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); 
alpha-1-antitrypsin gene control region which is active in liver (Kelsey 
et al., 1987, Genes and Devel. 1:161-171); beta-globin gene control region 
which is active in myeloid cells (Magram et al., 1985, Nature 315:338-340; 
Kollias et al., 1986, Cell 46:89-94); myelin basic protein gene control 
region which is active in oligodendrocyte cells in the brain (Readhead et 
al., 1987, Cell 48:703-712); myosin light chain-2 gene control region 
which is active in skeletal muscle (Shani, 1985, Nature 314:283-286); and 
gonadotropic releasing hormone gene control region which is active in the 
hypothalamus (Mason et al., 1986, Science 234:1372-1378). Promoters 
isolated from the genome of viruses that grow in mammalian cells, (e.g., 
RSV, vaccinia virus 7.5K, SV40, HSV, adenoviruses MLP, MMTV LTR and CMV 
promoters) may be used, as well as promoters produced by recombinant DNA 
or synthetic techniques. 
In some instances, the promoter elements may be constitutive or inducible 
promoters and can be used under the appropriate conditions to direct high 
level or regulated expression of the gene of interest. Expression of genes 
under the control of constitutive promoters does not require the presence 
of a specific substrate to induce gene expression and will occur under all 
conditions of cell growth. In contrast, expression of genes controlled by 
inducible promoters is responsive to the presence or absence of an 
inducing agent. 
Specific initiation signals are also required for sufficient translation of 
inserted protein coding sequences. These signals include the ATG 
initiation codon and adjacent sequences. In cases where the entire coding 
sequence, including the initiation codon and adjacent sequences are 
inserted into the appropriate expression vectors, no additional 
translational control signals may be needed. However, in cases where only 
a portion of the coding sequence is inserted, exogenous translational 
control signals, including the ATG initiation codon must be provided. 
Furthermore, the initiation codon must be in phase with the reading frame 
of the protein coding sequences to ensure translation of the entire 
insert. These exogenous translational control signals and initiation 
codons can be of a variety of origins, both natural and synthetic. The 
efficiency and control of expression may be enhanced by the inclusion of 
transcription attenuation sequences, enhancer elements, etc. 
In addition to DNA sequences encoding therapeutic proteins of interest, the 
scope of the present invention includes the use of ribozymes or antisense 
DNA molecules that may be transferred into the mammalian repair cells. 
Such ribozymes and antisense molecules may be used to inhibit the 
translation of RNA encoding proteins of genes that inhibit a disease 
process or the wound healing process thereby allowing tissue repair to 
take place. 
The expression of antisense RNA molecules will act to directly block the 
translation of mRNA by binding to targeted mRNA and preventing protein 
translation. The expression of ribozymes, which are enzymatic RNA 
molecules capable of catalyzing the specific cleavage of RNA may also be 
used to block protein translation. The mechanism of ribozyme action 
involves sequence specific hybridization of the ribozyme molecule to 
complementary target RNA, followed by a endonucleolytic cleavage. Within 
the scope of the invention are engineered hammerhead motif ribozyme 
molecules that specifically and efficiently catalyze endonucleolytic 
cleavage of RNA sequences. RNA molecules may be generated by transcription 
of DNA sequences encoding the RNA molecule. 
It is also within the scope of the invention that multiple genes, combined 
on a single genetic construct under control of one or more promoters, or 
prepared as separate constructs of the same or different types may be 
used. Thus, an almost endless combination of different genes and genetic 
constructs may be employed. Certain gene combinations may be designed to, 
or their use may otherwise result in, achieving synergistic effects on 
cell stimulation and regeneration, any and all such combinations are 
intended to fall within the scope of the present invention. Indeed, many 
synergistic effects have been described in the scientific literature, so 
that one of ordinary sill in the art would readily be able to identify 
likely synergistic gene combinations, or even gene-protein combinations. 
5.1.3 PREATION OF THE GENE ACTIVATED MATRICES 
In preferred embodiments, matrix or implant material is contacted with the 
DNA encoding a therapeutic product of interest by soaking the matrix 
material in a recombinant DNA stock solution. The amount of DNA, and the 
amount of contact time required for incorporation of the DNA into the 
matrix, will depend on the type of matrix used and can be readily 
determined by one of ordinary skill in the art without undue 
experimentation. Alternatively, the DNA may be encapsulated within a 
matrix of synthetic polymers, such as, for example, block copolymers of 
polyactic-polyglycolic acid (See Langer and Folkman, 1976 Nature, 
263:797-800 which is incorporated herein by reference). Again, these 
parameters can be readily determined by one of ordinary skill in the art 
without undue experimentation. For example, the amount of DNA construct 
that is applied to the matrix will be determined considering various 
biological and medical factors. One would take into consideration the 
particular gene, the matrix, the site of the wound, the mammalian host's 
age, sex and diet and any further clinical factors that may effect wound 
healing such as the serum levels of various factors and hormones. 
In additional embodiments of the invention compositions of both biological 
and synthetic matrices and DNA may be lyophilized together to form a dry 
pharmaceutical powder. The gene activated matrix may be rehydrated prior 
to implantation in the body, or alternatively, the gene activated matrix 
may become naturally rehydrated when placed in the body. 
In some instances medical devices such as implants, sutures, wound 
dressings, etc. may be coated with the nucleic acid compositions of the 
invention using conventional coating techniques as are well known in the 
art. Such methods include, by way of example and not limitation, dipping 
the device in the nucleic acid composition, brushing the device with the 
nucleic acid composition and/or spraying the device with the aerosol 
nucleic acid compositions of the invention. The devide is then dried, 
either at room temperature or with the aid of a drying oven, optionally at 
reduced pressure. A preferred method for coating sutures is provided in 
the examples. 
For sutures coated with a polymeric matrix containing plasmid DNA, 
applicants have discovered that applying a coating composition containing 
a total of about 0.01 to 10 mg plasmid DNA and preferably about 1 to 5 mg 
plasmid DNA, to a 70 cm length of suture using about 5 to 100, preferably 
about 5 to 50, and more preferably about 15 to 30 coating applications 
yields a therapeutically effective and uniform coating. 
In a particularly preferred embodiment, the invention provides coated 
sutures, especially sutures coated with a polymeric matrix containing 
nucleic acids encoding therapeutic proteins that stimulate wound healing 
in vivo. 
Sutures which may be coated in accordance with the methods and compositions 
of the present invention include any suture of natural or synthetic 
origin. Typical suture materials include, by way of example and not 
limitation, silk; cotton; linen; polyolefins such as polyethylene and 
polypropylene; polyesters such as polyethylene terephthalate; homopolymers 
and copolymers of hydroxycarboxylic acid esters; collagen (plain or 
chromicized); catgut (plain or chromicized); and suture-substitutes such 
as cyanoacrylates. The sutures may take any convenient form such as braids 
or twists, and may have a wide range of sizes as are commonly employed in 
the art. 
The advantages of coated sutures, especially sutures coated with a 
polymeric matrix containing nucleic acids encoding therapeutic proteins 
that stimulate wound healing cover virtually every field of surgical use 
in humans and animals. 
5.2. USES OF THE GENE ACTIVATED MATRIX 
The invention is applicable to a wide variety of wound healing situations 
in human medicine. These include, but are not limited to, bone repair, 
tendon repair, ligament, repair, blood vessel repair, skeletal muscle 
repair, and skin repair. For example, using the gene activated matrix 
technology, cytokine growth factors produced by transfected repair cells 
will influence other cells in the wound, through binding of cell surface 
signaling receptors, thereby stimulating and amplifying the cascade of 
physiological events normally associated with the process of wound 
healing. The end result is the augmentation of tissue repair and 
regeneration. 
The method of the invention also is useful when the clinical goal is to 
block a disease process, thereby allowing natural tissue healing to take 
place, or when the goal is to replace a genetically defective protein 
function. 
Wounds may arise from traumatic injury, or alternatively, from tissue 
damage either induced by, or resulting from, a surgical procedure. The 
gene activated matrix of the invention can be transferred to the patient 
using various techniques. For example, matrices can be transferred 
directly to the site of the wound by the hand of the physician, either as 
a therapeutic implant or as a coated device (e.g., suture, stent, coated 
implant, etc.). Matrices can be topically administered, either as placed 
surgically in a normal tissue site in order to treat diseased tissue some 
distance away. 
The process of wound healing is a coordinated sequence of events which 
includes, hemorrhage, clot formation, dissolution of the clot with 
concurrent removal of damaged tissue, and deposition of granulation tissue 
as initial repair material. The granulation tissue is a mixture of 
fibroblasts and capillary blood vessels. The wound healing process 
involves diverse cell populations including endothelial cells, stem cells, 
macrophages and fibroblasts. The regulatory factors involved in wound 
repair are known to include systemic hormones, cytokines, growth factors, 
extracellular matrix proteins and other proteins that regulate growth and 
differentiation. 
The DNA transfer methods and matrix compositions of the present invention 
will have a wide range of applications as a drug delivery method for 
stimulating tissue repair and regeneration in a variety of different types 
of tissues. These include but are not limited to bone repair, skin repair, 
connective tissue repair, organ regeneration, or regulation of 
vasculogenesis and/or angiogenesis. The use of gene activated matrices may 
also be used to treat patients with impaired healing capacity resulting 
from, for example, the effects of aging or diabetes. The matrices may also 
be used for treatment of wounds that heal slowly due to natural reasons, 
e.g., in the elderly, and those who do not respond to existing therapies, 
such as in those individuals with chronic skin wounds. 
One important feature of the present invention is that the formation of 
scar tissue at the site of the wound may be regulated by the selective use 
of gene activated matrices. The formation of scar tissue may be regulated 
by controlling the levels of therapeutic protein expressed. In instances, 
such as the treatment of burns or connective tissue damage it is 
especially desirable to inhibit the formation of scar tissue. 
The methods of the present invention include the grafting or 
transplantation of the matrices containing the DNA of interest into the 
host. Procedures for transplanting the matrices may include surgical 
placement, or injection, of the matrices into the host. In instances where 
the matrices are to be injected, the matrices are drawn up into a syringe 
and injected into a patient at the site of the wound. Multiple injections 
may be made in the area of the wound. Alternatively, the matrices may be 
surgically placed at the site of the wound. The amount of matrices needed 
to achieve the purpose of the present invention i.e. stimulation of wound 
repair and regeneration, is variable depending on the size, age and weight 
of the host. 
It is an essential feature of the invention that whenever a gene activated 
matrix is transferred to the host, whether by injection or surgery, that 
the local tissue damage be sufficient enough to induce the wound healing 
process. This is a necessary prerequisite for induction of migration and 
proliferation of the targeted mammalian repair cells to the site of the 
gene activated matrix. 
Specific embodiments are described in the sections that follow. 
5.3. BONE REGENERATION 
Bone has a substantial capacity to regenerate following fracture. The 
complex but ordered fracture repair sequence includes hemostasis, clot 
dissolution, granulation tissue ingrowth, formation of a callus, and 
remodeling of the callus to an optimized structure (A. W. Ham., 1930, J. 
Bone Joint Surg. 12, 827-844). Cells participating in this process include 
platelets, inflammatory cells, fibroblasts, endothelial cells, pericytes, 
osteoclasts, and osteogenic progenitors. Recently, several peptide growth 
and differentiation factors have been identified that appear to control 
cellular events associated with bone formation and repair (Erlebacher, A., 
et al., 1995, Cell 80, 371-378). Bone morphogenetic proteins (BMPs), for 
example, are soluble extracellular factors that control osteogenic cell 
fate: BMP genes are normally expressed by cultured fetal osteoblasts 
(Harris, S. E., et al., 1994, J. Bone Min. Res. 9, 389-394) and by 
osteoblasts during mouse embryo skeletogenesis (Lyons, K. M., et al., 
1989, Genes Dev. 3, 1657-1668; Lyons, K. M., et al., 1990, Development 
190, 833-844; Jones, M. C., et al., 1991, Development 111, 531-542), 
recombinant BMP proteins initiate cartilage and bone progenitor cell 
differentiation (Yamaguchi, A., et al., 1991, J. Cell Biol. 113, 681-687; 
Ahrens, M., et al., 1993, J. Bone Min. Res. 12, 871-880; Gitelman, S. E., 
et al., 1994, J. Cell Biol. 126, 1595-1609; Rosen, V., et al., 1994, J. 
Cell Biol. 127, 1755-1766), delivery of recombinant BMPs induce a bone 
formation sequence similar to endochondral bone formation (Wozney, J. M., 
1992, Mol. Reprod. Dev. 32, 160-167; Reddi, A. H., 1994, Curr. Opin. 
Genet. Dev. 4, 737-744), and BMP-4 gene expression is unregulated early in 
the process of fracture repair (Nakase, T., et al., 1994, J. Bone Min. 
Res. 9, 651-659). Osteogenic protein-1, a member of a family of molecules 
related to the BMPs (Ozkaynak, E., et al., 1990, EMBO J. 9, 2085-2093), is 
capable of similar effects in vitro and in vivo (Sampath, T. K., et al., 
1992, J. Biol. Chem. 267, 20352-20362; Cook, S. D., et al., (1994) J. Bone 
Joint Surg. 76-A, 827-838). TGF-.beta. has also been shown to stimulate 
cartilage and bone formation in vivo (Centrella, M., et al., 1994, 
Endocrine Rev. 15, 27-38; Sumner, D. R., et al., 1995, J. Bone Joint Surg. 
77A, 1135-1147). Finally, parathyroid hormone (PTH) is an 84 amino acid 
hormone that raises the plasma and extracellular fluid Ca.sup.+2 
concentration. In skeletal tissues, intermittent administration of a PTH 
fragment-possessing the structural requirements for biological activity 
(aa 1-34) produces a true anabolic effect: numerous in vivo and in vitro 
studies provide strong evidence that PTH1-34 administration in animals 
(including rats) results in uncoupled, high-quality bone formation due to 
a combined inhibitory effect on osteoclasts and stimulatory effect on 
osteogenic cells (Dempster, D. W., et al., 1993, Endocrine Rev. 14, 
690-709). The PTH1-34 peptide is known to interact synergistically with 
BMP-4, which up-regulates the expression of functional cell surface PTH 
receptors in differentiating osteoblasts in vitro (Ahrens, M., et al., 
1993, J. Bone Min. Res. 12, 871-880). 
As recombinant proteins, peptide growth and differentiation factors such as 
BMP and TGF-.beta.1 represent promising therapeutic alternatives for 
fracture repair (Wozney, J. M., 1992, Mol. Reprod. Dev. 32, 160-167; 
Reddi, A. H., 1994, Curr. Opin. Genet. Dev. 4, 737-744; Centrella, M., et 
al., 1994, Endocrine Rev. 15, 27-38; Sumner, D. R., et al., 1995 J. Bone 
Joint Surg. 77-A, 1135-1147). However, relatively large doses (microgram 
amounts) are required to stimulate significant new bone formation in 
animals, raising the concern that future human therapies may be expensive 
and may possess an increased risk of toxicity. 
In an embodiment of the invention, gene activated matrices are surgically 
implanted into a 5 mm osteomy site in the rat, a model of a complex, 
non-healing fracture in humans. The present inventors have found that gene 
transfer to repair cells in the osteotomy gap could be readily achieved. 
Defects in the process of bone repair and regeneration are associated with 
significant complications in clinical orthopaedic practice, for example, 
fibrous non-union following bone fracture, implant interface failures and 
large allograft failures. Many complex fractures are currently treated 
using autografts but this technique is not effective and is associated 
with complications. 
Naturally, any new technique designed to stimulate bone repair would be a 
valuable tool in treating bone fractures. A significant portion of 
fractured bones are still treated by casting, allowing natural mechanisms 
to effect wound repair. Although there have been advances in fracture 
treatment in recent years, including improved devices, the development of 
new processes to stimulate, or complement, the wound repair mechanisms 
would represent significant progress in this area. 
The present invention may be used to transfer a bone growth gene to promote 
fracture repair. Other important aspects of this technology include the 
use of gene transfer to treat patents with "weak bones", such as in 
diseases like osteoporosis; to improve poor healing which may arise for 
unknown reasons, e.g., fibrous non-union; to promote implant integration 
and the function of artificial joints; to stimulate healing of other 
skeletal tissues such as Achilles tendon; and as an adjuvant to repair 
large defects. 
Bone tissue is known to have the capacity for repair and regeneration and 
there is a certain understanding of the cellular and molecular basis of 
these processes. The initiation of new bone formation involves the 
commitment, clonal expansion, and differentiation of repair cells. Once 
initiated, bone formation is promoted by a variety of polypeptide growth 
factors. Newly formed bone is then maintained by a series of local and 
systemic growth and differentiation factors. 
Several bone morphogenetic protein genes have now been cloned (Wozney et 
al., 1988; Rosen et al. 1989, Connect. Tissue Res., 20:313:319; summarized 
in Alper, 1994) and this work has established BMPs as members of the 
transforming growth factor-.beta. (TGF-.beta.) superfamily based on DNA 
sequence homologies. The cloning of distinct BMP genes has led to the 
designation of individual BMP genes and proteins as BMP-1 through at least 
BMP-8. BMPs 2-8 are generally thought to be osteogenic while BMP-1 may be 
a more generalized morphogen; Shimell et al., 1991, Cell, 67:469-481). 
BMP-3 is also called osteogen (Luyten et al., 1989, J. Biol. Chem., 
264:13377-13380) and BMP-7 is also called OP-1 (Ozkaynak et al., 1990, 
EMBO J., 9:2085-2093). TGFs and BMPs each act on cells via complex, 
tissue-specific interactions with families of cell surface receptors 
(Roberts & Sporn, 1989, M. B. Sporn and A. B. Roberts, Eds., 
Springer-Verlag, Heidelberg, 95 (Part 1); Aralkar et al., 1991). 
Transforming growth factors (TGFs) have also been shown to have a central 
role in regulating tissue healing by affecting cell proliferation, gene 
expression, and matrix protein synthesis (Roberts & Sporn, 1989, M. B. 
Sporn and A. B. Roberts, Eds., Springer-Verlag, Heidelberg, 95 (Part 1)). 
For example, TGF-.beta.1 and TGF-.beta.2 can initiate both chondrogenesis 
and osteogenesis (Joyce et al., 1990, J. Cell Biol., 110:195-2007; Izumi 
et al., 1992, J. Bone Min. Res., 7:115-11; Jingushi et al., 1992, J. 
Orthop. Res., 8:364-371). 
Other growth factors/hormones besides TGF and BMP can be used in the 
practice of the invention to influence new bone formation following 
fracture. For example, fibroblast growth factor injected into a rat 
fracture site (Jingushi et al., 1990) at multiple high doses (1.0. mg/50 
ml) resulted in a significant increase in cartilage tissue in the fracture 
gap, while lower doses had no effect. 
Calcium regulating hormones such as parathyroid hormone (PTH) may also be 
used in one aspect of the invention. PTH is an 84 amino acid 
calcium-regulated hormone whose principal function is to raise Ca.sup.+2 
concentration in plasma and extracellular fluid. Intact PTH was also shown 
to stimulate bone reabsorption in organ culture over 30 years ago, and the 
hormone is known to increase the number and activity of osteoclasts. 
Studies with the native hormone and with synthetic peptides have 
demonstrated that the amino terminus of the molecule (aa-1-34) contains 
the structural requirements for biological activity (Tregear et al., 1973; 
Hermann-Erlee et al., 1976, Endocrine Research Communications, 3:21-35; 
Riond, 1993, Clin. Sci., 85:223-228). 
In an embodiment of the invention the gene activated matrices are 
surgically implanted into the site of the bone fracture. Such surgical 
procedures may include direct injection of a GAM preparation into the 
fracture site, the surgical repair of a complex fracture, or arthroscopic 
surgery. In instances where the gene activated matrices are being used to 
repair fractured bone, the mammalian repair cells will naturally migrate 
and proliferate at the site of bone damage. 
The present inventors have surprisingly found that gene transfer into 
repair cells in the regenerating tissue in the osteotomy gap could be 
readily achieved. Currently, the preferred methods for achieving gene 
transfer generally involve using a fibrous collagen implant material 
soaked in a solution of DNA shortly before being placed in the site in 
which one desires to promote bone growth or using a preparation of plasmid 
DNA encapsulated in a synthetic matrix such as a block copolymer of PLGA. 
As the studies presented herein show, the implant material facilitates the 
targeted uptake of exogenous plasmid constructs by cells in the osteotomy 
gap, which clearly participate in bone regeneration/repair. The 
transgenes, following cellular uptake, direct the expression of 
recombinant polypeptides, as evidenced by the in vivo expression of 
functional marker gene products. 
Further studies are presented herein demonstrating that the transfer of an 
osteotropic gene results in cellular expression of a recombinant 
osteotropic molecule, which expression is directly associated with 
stimulation of new bone formation. Specifically, a gene transfer vector 
coding for BMP-4 and a gene transfer vector encoding a fragment of human 
PTH1-34, alone and in combination, will stimulate new bone formation. 
After considering a relatively large number of candidate genes, a gene 
transfer vector coding for a fragment of human parathyroid hormone, 
hPTH1-34, will stimulate new bone formulation in Sprague-Dawley rats, 
indicating that the human peptide can efficiently bind the PTH/PTHrP 
receptor on the rat osteoblast cell surface. 
5.4. SOFT TISSUES 
The present invention may also be used to stimulate the growth or 
regeneration of soft tissues such as ligament, tendon, cartilage and skin. 
Skeletal connective tissue damage due to traumatic injury may be treated 
using matrices containing genes encoding a variety of growth factors. 
Connective tissue normally consists of cells and extracellular matrix 
organized in a characteristic tissue architecture. Tissue wounding can 
disrupt this architecture and stimulate a wound healing response. The 
methods of the present invention are particularly well suited for 
stimulation of growth and regeneration of connective tissue as it is 
important that the injured tissue regenerate without the formation of scar 
tissue as scar tissue can interfere the normal mechanical function of 
connective tissue. 
Various growth factors may be used to promote soft tissue repair. These 
include, but are not limited to, members of the TGF-.beta. superfamily 
(e.g., TGF-.beta. itself), which stimulates expression of genes coding for 
extracellular matrix proteins, and other cytokines such as EGF and PDGF. 
Examples of other genes that may be used include (a) cytokines such as the 
peptide growth and differentiation factors, interleukines, chemokines, 
interferons, colony stimulating factors; (b) angiogenic factors such as 
FGF and VEGF; (c) extracellular matrix proteins such as collagen, laminin, 
and fibronectin; (d) the family of cell adhesion molecules (e.g., the 
integrins, selectins, Ig family members such as N-CAM and L1, and 
cadherins); (e) cell surface cytokine signaling receptors such as the type 
I and type II TGF-.beta. receptors and the FGF receptors; (f) 
non-signaling co-receptors such as betaglycan and syndecan; (g) the family 
of signal transducing kinases; (h) cytoskeletal proteins such as talin and 
vinculin; (i) cytokine binding proteins such as the family of latent 
TGF-.beta. binding proteins; and (j) nuclear trans acting proteins such as 
transcription factors. 
Once formed, such matrices, may then be placed in the host mammal in the 
area of the connective tissue wound. The gene activated matrices may be 
injected directly into the area of connective tissue injury. 
Alternatively, surgical techniques, such as arthroscopic surgery, may be 
used to deliver the matrices to the area of the connective tissue wound. 
5.5. ORGAN REGENERATION 
The present invention may also be used to stimulate the repair and 
regeneration of organ tissue. Organ damage due to traumatic injury, or 
surgery, may be treated using the methods of the present invention. In the 
case of liver, the liver may be damaged due to excessive alcohol 
consumption or due to infection with various types of infectious agents 
such as the hepatitis family of viruses. The kidney may likewise fail to 
function normally as a result of damage resulting from kidney disease. 
Mucous membranes of the esophagus, stomach or duodenum may contain 
ulcerations caused by acid and pepsin in gastric juices. The ulcerations 
may also arise from colonization of gastric mucosal cells with 
Helicobacter pylori bacteria. These organs and diseases serve only as 
examples, indeed the methods of the invention may be used to treat 
diseases, or to stimulate organ regeneration in any organ of the body. 
Matrices containing DNA encoding cytokines which stimulate proliferation 
and differentiation of cells, and/or regulate tissue morphogenesis, may be 
transplanted to the appropriate organ site. Such factors may include but 
are not limited to, the transforming growth factor family of proteins, 
platelet derived growth factor (PDGF), insulin like growth factor (IGF) 
and fibroblast growth factory (FGF). In some instances it may be useful to 
express growth factors and/or cytokines that stimulate the proliferation 
of cell types specific for a given organ, i.e., hepatocytes, kidney or 
cardiac cells, etc. For example, hepatocyte growth factor may be expressed 
to stimulate the wound healing process in the liver. For treatment of 
ulcers, resulting from Helicobacter infection, the gene activated matrices 
may contain DNA encoding anti-microbial proteins. 
The gene activated matrices of the invention can be surgically implanted 
into the organ that is to be treated. Alternatively, laproscopic surgical 
procedures may be utilized to transfer the gene activated matrices into 
the body. In cases where the treatment is in response to tissue injury, 
the natural wound healing process will stimulate the migration and 
proliferation of the repair cells to the transplanted matrices. 
Alternatively, where the gene activated matrices are transferred to organs 
which have not been injured, for example, where matrices are implanted to 
express therapeutic proteins not involved in wound healing, the wound 
healing process can be stimulated by induction of tissue injury. 
5.6. REGULATION OF ANGIOGENESIS 
The present invention may also be used to regulate the formation and 
spreading of blood vessels, or vasculogenesis and angiogenesis, 
respectively. Both these physiological processes play an important role in 
wound healing and organ regeneration. 
Initially, at the site of a wound, granulation tissue which is a mixture of 
collagen, matrix and blood vessels, is deposited and provides wound 
strength during tissue repair. The formation of new blood vessels involves 
the proliferation, migration and infiltration of vascular endothelial 
cells, and is known to be regulated by a variety of polypeptide growth 
factors. Several polypeptides with endothelial cell growth promoting 
activity have been identified, including acidic and basic fibroblastic 
growth factors (FGF), vascular endothelial growth factor (VEGF), and 
placental derived growth factor (PDGF). 
To stimulate the formation and spreading of blood vessels, DNA encoding 
such growth factors may be incorporated into matrices and these matrices 
may be implanted into the host. In some instances, it may be necessary to 
induce the wound healing process through tissue injury. 
It may be desirable to inhibit the proliferation of blood vessel formation, 
such as in angiogenesis associated with the growth of solid tumors which 
rely on vascularization for growth. Tumor angiogenesis may be inhibited 
through the transfer of DNA's encoding negative inhibitors of 
angiogenesis, such as thrombospondin or angiostatin. In specific 
embodiments of the invention, DNA encoding, for example, thrombospondin or 
angiostatin, may be incorporated into a matrix followed by the implanting 
of the matrix into a patient at the site of the tumor. 
5.7. REPAIR OF THE SKIN 
The present invention may also be used to stimulate the growth and repair 
of skin tissue. In wounds which involve injury to areas of the skin, and 
particularly in the case of massive burns, it is important that the skin 
grow very rapidly in order to prevent infections, reduce fluid loss, and 
reduce the area of potential scarring. Skin damage resulting from burns, 
punctures, cuts and/or abrasions may be treated using the gene activated 
matrices of the present invention. Skin disorders such as psoriasis, 
atopic dermatitis or skin damage arising from fungal, bacterial and viral 
infections or treatment of skin cancers such as melanoma, may also be 
treated using the methods of the invention. 
Matrices containing DNA encoding cytokines which stimulate proliferation 
and differentiation of cells of the skin, including central basal stem 
cells, keratinocytes, melanoytes, Langerhans cells and Merkel cells may be 
used to treat skin injuries and disorders. The gene activated matrices 
serve two functions, the protection of the wound from infection and 
dehydration and supplying the DNA for uptake by repair cells. The gene 
activated matrices of the invention may include dermal patches, cadaver 
skin, band-aids, gauze pads, collagen lattices such as those disclosed in 
U.S. Pat. No. 4,505,266 or U.S. Pat. No. 4,485,097, topical creams or 
gels. Prior to the application of the matrices to the wound site, damaged 
skin or devitalized tissue may be removed. The DNA to be incorporated into 
the matrices may encode a variety of different growth factors including 
keratinocyte-growth-factor (KGF) or epidermal growth factor (EGF). DNA 
encoding IL-1 which has been shown to be a potent inducer of epithelial 
cell migration and proliferation as part of the healing process may also 
be incorporated into the matrices of the invention. 
6. EXAMPLE: IMPLANT MATERIAL FOR USE IN BONE GENE TRANSFER 
Various implant materials may be used for transferring genes into the site 
of bone repair and/or regeneration in vivo. These materials are soaked in 
a solution containing the DNA or gene that is to be transferred to the 
bone regrowth site. Alternatively, DNA may be incorporated into the matrix 
as a preferred method of making. 
One particular example of a suitable material is fibrous collagen, which 
may be lyophilized following extraction and partial purification from 
tissue and then sterilized. Another particularly preferred collagen is 
type II collagen, with the most particularly preferred collagen being 
either recombinant type II collagen, or mineralized type II collagen. 
Prior to placement in osteotomy sites, implant materials are soaked in 
solutions of DNA (or virus) under sterile conditions. The soaking may be 
for any appropriate and convenient period, e.g., from 6 minutes to 
over-night. The DNA (e.g., plasmid) solution will be a sterile aqueous 
solution, such as sterile water or an acceptable buffer, with the 
concentration generally being about 0.5-1.0 mg/ml. Currently preferred 
plasmids are those such as pGL2 (Promega), pSV40.beta.-gal, pAd.CMVlacZ, 
and pcDNA3. 
7. EXAMPLE: IN VIVO PROTEIN DETECTION FOLLOWING TRANSGENE EXPRESSION 
7.1. .beta.-GALACTOSIDASE TRANSGENE 
Bacterial .beta.-galactosidase can be detected immunohistochemically. 
Osteotomy tissue specimens were fixed in Bouins fixative, demineralized, 
and then split in half along the longitudinal plane. One-half of each 
specimen was embedded in paraffin for subsequent immunohistochemical 
identification of the bacterial .beta.-galactosidase protein. 
For immunohistochemistry, cross-Sections (2-3 mm thick) were transferred to 
poly-L-Lysine coated microscope slides and fixed in acetone at 0.degree. 
C. for at least 20 min. Sections were rehydrated in PBS. Endogenous 
peroxidase activity was quenched by immersion of tissue sections in 0.1% 
hydrogen peroxide (in 95% methanol) at room temperature for 10 min, and 
quenched sections were washed 3x in PBS. In some cases, sectioned 
calvariae were demineralized by immersion in 4% EDTA, 5% polyvinyl 
pyrrolidone, and 7% sucrose, pH 7.4, for 24 h at 4.degree. C. 
Demineralized sections were washed 3x before application for antibodies. 
Primary antibodies were used without dilution in the form of hybridoma 
supernatant. Purified antibodies were applied to tissue sections at a 
concentration of 5 mg/ml. Primary antibodies were detected with 
biotinylated rabbit antimouse IgG and peroxidase conjugated streptavidin 
(Zymed Histostain-SPkit). After peroxidase staining, sections were 
counterstained with hematoxylin. 
Bacterial .beta.-gal was also detected by substrate utilization assays 
using commercially available kits (e.g., Promega) according to the 
manufacturers' instructions. 
7.2. LUCIFERASE TRANSGENE 
Luciferase was detected by substrate utilization assays using commercially 
available kits (e.g., Promega) according to the manufacturers' 
instructions. 
7.3. PTH TRANSGENES 
Recombinant PTH, such as hPTH1-34 peptide, was assayed in homogenates of 
osteotomy gap tissue, for example, using two commercially available 
radioimmunoassay kits according to the manufacturer's protocols (Nichols 
Institute Diagnostics, San Juan Capistrano, Calif.). 
One kit is the Intact PTH-Parathyroid Hormone 100T Kit. This 
radioimmunoassay utilizes an antibody to the carboxy terminus of the 
intact hormone, and this is used to measure endogenous levels of hormone 
in gap osteotomy tissue. This assay may be used to establish a baseline 
value PTH expression in the rat osteotomy model. 
The second kit is a two=site immunoradiometric kit for the measurement of 
rat PTH. This kit uses affinity purified antibodies specific for the amino 
terminus of the intact rat hormone (PTH1-34) and thus will measure 
endogenous PTH production as well as the recombinant protein. Previous 
studies have shown that these antibodies cross-react with human PTH and 
this are able to recognize recombinant molecules in vivo. 
Values obtained with kit #1 (antibody to the carboxy terminus) were 
subtracted from values obtained with kit #2 (antibody to the amino 
terminus) to obtain an accurate and sensitive measurements. The level of 
recombinant peptide was thus correlated with the degree of new bone 
formation. 
7.4. BMP TRANSGENE 
BMP proteins, such as the murine BMP-4 transgene peptide product, were 
detected immunohistochemically using a specific antibody that recognizes 
the HA epitope (Majmudar et al., 1991, J. Bone and Min. Res. 6:869-881), 
such as the monoclonal antibody available from Boehringer-Mannheim. 
Antibodies to BMP proteins themselves may also be used. Such antibodies, 
along with various immunoassay methods, are described in U.S. Pat. No. 
4,857,456, incorporated herein by reference. 
Osteotomy tissue specimens were fixed in Bouins fixative, demineralized, 
and then split in half along the longitudinal plane. One-half of each 
specimen was embedded in paraffin for subsequent immunohistochemical 
identification of the recombinant murine BMP-4 molecule. 
8. EXAMPLE: TRANSFER OF AN OSTEOTROPIC GENE STIMULATES BONE 
REGENERATION/REPAIR IN VIVO 
The following experiment was designed to investigate whether gene transfer 
could be employed to create transfected cells that constitutively express 
recombinant hPTH1-34 in vivo, and whether this transgene can stimulate 
bone formation. The rate of new bone formation was analyzed as follows. At 
necropsy the osteotomy site was carefully dissected for histomorphometric 
analysis. The A-P and M-L dimensions of the callus tissue are measured 
using calipers. Specimens were then immersion fixed in Bouins fixative, 
washed in ethanol, and demineralized in buffered formic acid. Plastic 
embedding of decalcified material was used because of the superior 
dimensional stability of methacrylate during sample preparation and 
sectioning. Tissue blocks were dehydrated in increasing alcohol 
concentrations and embedded. 5 mm thick sections were cut in the coronal 
plane using a Reichert Polycot microtome. Sections were prepared from 
midway through the width of the marrow cavity to guard against a sampling 
bias. Sections for light microscopy were stained using a modified 
Goldner's trichrome stain, to differentiate bone, osteoid, cartilage, and 
fibrous tissue. Sections were cover-slipped using Eukitt's mounting medium 
(Calibrated Instruments, Ardsley, N.Y.). Histomorphometric analyses were 
performed under brightfield using a Nikon Optiphot Research microscope. 
Standard point count stereology techniques using a 10 mm.times.10 mm 
eyepiece grid reticular. 
Total callus area was measured at 125x magnification as an index of the 
overall intensity of the healing reaction. Area fractions of bone, 
cartilage, and fibrous tissue were measured at 250x magnification to 
examine the relative contribution of each tissue to callus formation. 
Since the dimensions of the osteotomy gap reflect the baseline (time 0), a 
measurement of bone area at subsequent time intervals was used to indicate 
the rate of bone infill. Statistical significance was assessed using 
analysis of variance, with post-hoc comparisons between groups conducted 
using Tukey's studentized range test. 
In the 5 mm rat osteotomy model described above, it was found that PTH 
transgene expression can stimulate bone regeneration/repair in live 
animals. This is a particularly important finding as it is known that 
hPTH1-34 is a more powerful anabolic agent when given intermittently as 
opposed to continuously, and it is the continuous-type delivery that 
results from the gene transfer methods used here. 
9. EXAMPLE: DIRECT GENE TRANSFER INTO REGENERATING BONE IN VIVO 
Gene activated matrices containing mammalian expression plasmid DNA were 
implanted into large segmental gaps created in the adult male femur. 
Implantation of gene-activated matrices containing beta-galactosidase or 
luciferase plasmids led to DNA uptake and functional enzyme expression by 
repair cells growing into the gap. Additionally, implantation of a gene 
activated matrix containing either a bone morphogenetic protein-4 plasmid 
or a plasmid coding for a fragment of parathyroid hormone (amino acids 
1-34) resulting in a biological response of new bone filling the gap. 
Finally, implantation of a two-plasmid gene-activated matrix encoding bone 
morphogenetic protein-4 and the parathyroid hormone fragment, which have 
been shown to act synergistically in vitro, caused new bone to form faster 
than with either factor alone. These studies demonstrate that for the 
first time that repair cells in bone can be genetically manipulated in 
vivo. While serving as a useful tool to study the biology of repair 
fibroblasts and the wound healing response, the gene activated matrix of 
the present invention also has wide therapeutic utility. 
9.1. MATERIALS AND METHODS 
9.1.1. MAMMALIAN HOST MODEL 
To create a 5 mm osteotomy, four 1.2 mm diameter pins were screwed into the 
femoral diaphysis of normal adult Sprague-Dawley rats under general 
anesthesia and with constant irrigation. A surgical template guided 
parallel pin placement, which was confirmed by fluorography (pins were set 
3.5 mm from the edge of the fixator place and 2.5 mm apart). An external 
fixator place (30.times.10.times.5 mm) was then secured on the pins. 
External fixator plates were fabricated with aluminum alloy on a CNC mill 
to ensure high tolerances. Prefabricated fasteners with associated 
lockwashers and threaded pins were made of stainless steel. All fixator 
parts were sterilized with ethylene oxide gas prior to surgery. 5 mm 
segmental defects were created at mid-shaft with a Hall Micro 100 
oscillating saw (Zimmer Inc., Warsaw, Ind.). Collagen sponges were placed 
and held in the osteotomy gap until surrounded by clotted blood; 
preliminary studies showed that this maneuver fixed the sponge with the 
osteotomy site. The skin incision was closed with staples. The fixator 
provided the necessary stability so that the mammalian host's ambulation 
was unlimited for a several week period. 
9.1.2. IMMUNOHISTOCHEMISTRY 
Tissues were prepared for light microscopy and immunohistochemistry was 
performed as described (Wong et al., 1992, J. Biol. Chem. 267: 5592-5598). 
Histology sections were incubated with a commercially available 
anti-.beta.-gal antibody (1:200 dilution, 5 Prime.fwdarw.3 Prime) and with 
a commercially available anti-HA.11 polyclonal antibody (1:500 dilution, 
BAbCO). 
9.1.3. LUCIFERASE AND .beta.-gal ENZYME ASSAYS 
Luciferase and .beta.-gal activity was determined using the Luciferase 
Assay System (Promega) and .beta.-galactosidase Enzyme Assay System 
(Promega) according to protocols supplied by the manufacturer. 
9.1.4. PGAM1 EXPRESSION PLASMID 
To assemble pGAM1, mRNA was prepared from day 13.5 p.c. CD-1 mouse embryos 
using kit reagents and protocols (Poly AT Tract mRNA Isolation System I, 
Promega). An aliquot of mRNA was used to generate cDNA using commercial 
reagents (Reverse Transcriptase System, Promega). A full length mouse 
BMP-4 cDNA coding sequence was generated by the polymerase chain reaction 
(PCR) using the following conditions: 94.degree. C., 4 min., 1 cycle; 
94.degree. C., 1 min., 65.degree. C., 1 min., 72.degree. C., 1 min., 30 
cycles; 72.degree. C., 8 min., 1 cycle. The sequence of the PCR primers 
was based on the known mouse BMP-4 sequence (GenBank): upstream primer-5' 
CCATGATTCCTGGTAACCGAATGCTG 3'; downstream primer-5' CTCAGCGGCATCCGCACCCCTC 
3'. A single PCR product of the expected size (1.3 kb) was purified by 
agarose gel electrophoresis and cloned into the TA cloning vector 
(Invitrogen). The 5' end of the BMP-4 insert was further modified (PCR) by 
addition of a 27 nucleotide sequence that codes for the HA epitope, and 
the BMP-4 insert was cloned into the pcDNA3 expression vector 
(InVitrogen). Plasmid DNA was prepared and sequenced (both strands) to 
ensure the orientation and integrity of the BMP-4 insert. 
The pGAM1 plasmid was expressed using an in vitro transcription and 
translation kit (TNT T7 Coupled Reticulocyte Lysate System, Promega) 
according to protocols supplied by the manufacturer. Protein 
radiolabeling, immunoprecipitation, sample preparation and SDS-PAGE, 
autoradiography, transient transfection, and Western analysis were 
performed as described (Yin et al., 1995, J. Biol. Chem. 270:10147-10160). 
9.1.5. pGAM2 EXPRESSION PLASMID 
Human parathyroid hormone cDNA fragments encoding amino acids preprol-34 
were generated by PCR. The sequence of the PCR primers was based on known 
human PTH sequence (GenBank): upstream primer-5' 
GCGGATCCGCGATGATACCTGCAAAAGACATG 3'; downstream primer-5' 
GCGGATCCGCGTCAAAAATTGTGCACATCC 3'. This primer pair created BamHI sites at 
both ends of the PCR fragment. The fragment was digested with BamHI and 
ligated into a BamHI cloning site in the PLJ retrovirus vector (Wilson et 
al., 1992, Endocrinol. 130: 2947-2954). A clone with the insert in the 
coding orientation (pGAM2) eventually was isolated and characterized by 
DNA sequence analysis. 
To generate retroviral stocks, the .phi. CRIP packaging cell line (Wilson, 
J. M., et al., 1992, Endocrinology 130:2947-2954) was transfected with 10 
.mu.g of recombinant vector DNA using the calcium phosphate method. After 
an overnight incubation, culture medium (Dulbecco's Modified Eagle's 
Medium, supplemented with 10% fetal bovine serum, penicillin (100 
units/ml), and streptomycin (100 mg/ml) (all reagents from Gibco-BRL Life 
Technologies, Inc.) containing retrovirion particles was harvested and 
applied to cultured Rat-1 cells. Independent clones of successfully 
transduced Rat-1 cells were obtained by standard infection and selection 
procedures. Briefly, cultured Rat-1 cells were grown to confluence, split 
1:10, and selected in G418 (1 mg/ml. Gibco-BRL Life Technologies, Inc.). 
In some instances, antibiotic-resistant colonies were pooled into a single 
culture. In other instances, single colonies of resistant cells were 
maintained. Similar methods were used to generated clones of Rat-1 cells 
transduced with the BAGT retrovirus, which encodes the bacterial b-gal 
enzyme. 
The hPTH1-34 concentration in cell culture media was estimated using a 
commercial radioimmunoassay kit (INS-PTH, Nichols) and according to the 
manufacturer's protocol. The biological activity of the peptide encoded by 
pGAM2 was evaluated as described (McCauley, et al., 1994, Mol. Cell. 
Endocrinol. 101: 331-336). 
9.1.6. PREATION OF GENE ACTIVATED COLLAGEN SPONGES 
For each osteotomy gap, lyophilized bovine tracheal collagen (10 mg, 
Sigma), was thoroughly wetted in a sterile solution of 0.5-1.0 mg plasmid 
DNA and allowed to incubate for 1-16 hours at 4.degree. C. prior to 
implantation. 
9.1.7. RADIOGRAPHY 
Weekly plain film radiographs (posterior-anterior view) were obtained while 
mammalian hosts were awake using a portable X-ray unit (GE, model 100). 
The exposure was 1/10 sec at 57 kV and 15 ma. 
9.2. RESULTS 
9.2.1. OSTEOTOMY MODEL 
Our model system employed a 5 mm mid-shaft osteotomy in the adult rat 
femur. The osteotomy gap was stabilized by a four-pin external fixator. 
Whereas osteotomy repair in the rat is completed by 9 weeks post-surgery, 
the manner of repair depends on the size of the gap: a 2 mm gap heals by 
bony union, but a 5 mm gap heals by fibrous nonunion (Rouleau, J. P., et 
al., Trans. Ortho. Res. Soc. 20:). Controlled mammalian hosts maintained 
for up to 13 weeks post-surgery confirmed the observation that 5 mm gaps 
typically heal by fibrous nonunion. Weekly plain film radiography and 
histology (FIG. 1A-D) demonstrated that bone did not form in mammalian 
hosts that received either a 5 mm osteotomy alone (n=3), a 5 mm osteotomy 
plus a collagen sponge (n=10), or a 5 mm osteotomy plus a collagen sponge 
containing marker gene naked plasmid DNA (n=23). All 36 control gaps 
healed by deposition of fibrous tissue. Control femurs exhibited focal 
periosteal new bone formation (a complication of pin placement). A focal, 
transient inflammatory response (lymphocytes and macrophages) in gap 
tissues was also observed post-surgery. 
9.2.2. MARKER GENE STUDIES 
In a preliminary feasibility study, lacZ and .beta.-gal expression plasmid 
DNA were successfully transferred in vivo. 
The goal was to standardize the gene activated matrix preparation protocol 
and post-operative time course. A GAM encoding luciferase was placed in 
the osteotomy gap of one rat and a gene activated matrix encoding 
.beta.-gal was placed in the gap of a second animal. Three weeks later, 
gap homogenates (consisting a granulation tissue) were prepared after 
careful dissection of surrounding bone, cartilage, and skeletal muscle. 
Aliquots of each homogenate were evaluated for enzyme expression by 
substrate utilization assay. The expected enzyme activity was detected in 
each homogenate sample. Positive results were obtained in other 
experiments in which conditions varied (e.g., DNA dose, time to assay 
protein expression). 
9.2.3. BMP-4 GENE TRANSFER 
Having demonstrated that gap cells express functional enzymes following 
uptake of plasmid DNA from a matrix, we asked whether gene transfer could 
be used to modulate bone regeneration. We chose to overexpress BMP-4, an 
osteoinductive factor that normally is expressed by progenitor cells 
during fracture repair. A full length mouse BMP-4 CDNA was generated by 
PCR and subcloned into the pcDNA3 (Invitrogen) eukaryotic expression 
vector (FIG. 2). To specifically detect recombinant proteins, the 3' end 
of the BMP-4 coding sequence was modified by addition of a hemagglutinin 
(HA) epitope. Recombinant BMP-4 was expressed from this construct (pGAMI) 
using an in vitro transcription and translation protocol. 
Immunoprecipitation studies established the ability of the HA epitope to 
be recognized by an anti-HA polyclonal antibody. Biosynthesis of 
recombinant BMP-4 was evaluated following transient transfection of 
cultured 293T cells with PGAMI plasmid DNA. As demonstrated by 
immunoprecipitation, BMP-4 molecules were assembled into homodimers, 
secreted, and processed as expected. Taken together these results 
established that the HA-epitope was recognized by the anti-HA polyclonal 
antibody. 
Collagen sponges containing pGAMI DNA were placed in the gap of nine adult 
rats maintained for 4-24 weeks. In one mammalian host sacrificed 4 weeks 
post surgery, immunohistochemical studies using the anti-HA antibody 
demonstrated PGAMI expression by repair fibroblasts within the gap. This 
was significant, given that we did not observe false positive staining in 
a survey of gap tissue from thirteen control mammalian hosts. Microscopic 
foci of new bone, originating from both surgical margins, were also 
observed in the 4 week specimens. Consistent with a classic description of 
bone formation by autoinduction (Urist, 1965, Science 150:893-999), these 
foci consisted of bony plates surfaced by large cuboidal osteoblasts and 
supported by a cellular connective tissue composed of pleomorphic spindled 
fibroblasts and capillary vessels. In seven mammalian hosts sacrificed 
5-12 weeks post-surgery, the amount of radiographic new bone steadily 
increased (FIG. 3A), even though BMP-4 encoded by the transgene was not 
detectable by immunohistochemistry. Bridging, defined as new bone 
extending from the surgical margins across the osteotomy gap, typically 
was observed by 9 weeks. A ninth mammalian host survived without 
complication for 24 weeks post-surgery. Sufficient new bone formed by 18 
weeks to allow removal of the external fixator, and the mammalian host 
ambulated well for an additional 6 weeks (FIG. 3D). At sacrifice, the gap 
was filled with new bone undergoing active remodeling, with the exception 
of a thin strip of radiolucent tissue near the distal margin of the gap. 
Given that the mammalian host had successfully ambulated without fixation, 
this strip was assumed to be partially mineralized. Consistent with this 
hypothesis biomechanical testing (Frankenburg et al., 1994, Trans. Ortho. 
Res. Soc. 19:513), which demonstrated that the healed gap had essentially 
the same mechanical strength as the unoperated femur from the same 
mammalian host (6.3% difference, maximum torque test). The radiographic 
appearance of the contralateral (unoperated) femur was unchanged in all 
nine cases, implying that the effects of gene transfer and BMP-4 
overexpression were limited to the osteotomy gap. 
9.2.4. TRANSFER AND EXPRESSION OF A PLASMID COCKTAIL (BMP-4+PTH1-34) 
Bone regeneration normally is governed by multiple factors acting in a 
regulated sequence, and we wondered, therefore, if the expression of 
several anabolic factors would stimulate bone formation more powerfully 
than a single factor alone. To evaluate this hypothesis, we chose to 
deliver a two-plasmid GAM encoding BMP-4 plus a peptide fragment of 
parathyroid hormone (PTH). PTH is an 84 amino acid hormone that raises the 
plasma and extracellular fluid Ca.sup.+2 concentration. In skeletal 
tissues, the intermittent administration of a PTH fragment possessing the 
structural requirements for biological activity (aa 1-34) produces a true 
anabolic effect: numerous in vivo and in vitro studies provide strong 
evidence that PTH1-34 administration in mammalian hosts (including rats) 
results in uncoupled, high-quality bone formation due to a combined 
inhibitory effect on osteoclasts and stimulatory effect on osteogenic 
cells (Dempster et al., 1993, Endocrin Rev. 14:690-709). The PTH1-34 
peptide is known to interact synergistically with BMP-4, which 
up-regulates the expression of functional cell surface PTH receptors in 
differentiating osteoblasts (Ahrens et al., 1993, J. Bone Min. Res. 
12:871-880). 
A cDNA fragment encoding human PTH1-34 was generated by PCR. To establish 
its biological activity, the fragment was subcloned into the PLJ 
retroviral vector (Wilson et al., 1992, Endocrin, 130:2947-2954), 
generating the pGAM2 expression plasmid (FIG. 4A). A stock of 
replication-defective, recombinant retrovirus was prepared and applied to 
Rat-I cells in culture. Independent clones of transduced Rat-I cells were 
obtained, and stable integration and expression of retroviral DNA was 
demonstrated by Southern and Northern analyses. Radioimmunoassay was used 
to establish the concentration of human PTH 1-34 in conditioned media of 
individual clones. ROS 17/2.8 cells possess PTH cell surface receptors, 
which belong to the G protein-coupled receptor superfamily (Dempster et 
al., 1993, Endocrin. Rev. 14:690-709). Incubation of ROS 17/2.8 cells with 
aliquots of conditioned media from a stably transduced cell line 
(secreting &gt;2 pg/ml via radioimmunoassay) resulted in a 2.7-fold increase 
in CAMP response versus the control, a result that established that the 
secreted PTH1-34 peptide was biologically active. 
GAMs containing pGAM2 plasmid DNA alone stimulated bone (FIG. 4B) GAMs 
containing the BMP-4 and PTH1-34 expression plasmid DNAs together were 
then implanted in the osteotomy gap of an additional three mammalian 
hosts. Bridging was observed by 4 weeks in all three mammalian hosts (one 
mammalian host was sacrificed at this time for histology), and sufficient 
new bone had formed by 12 weeks post-implantation in the remaining 
mammalian hosts to allow removal of the external fixator (FIG. 5). Both 
mammalian hosts are ambulating well at the time of publication 15 and 26 
weeks post-implantation, respectively. Based on plain-film radiography, 
the effects of gene transfer and overexpression again appeared to be 
limited to the osteotomy gap. 
Subsequent to studies using a collagen sponge, it has also been shown that 
plasmid DNA could be delivered to cells in a sustained manner following 
encapsulation within a preparation of block co-polymers of 
polylactic-polyglycolic particles. The results demonstrate that cultured 
cells can be transfected by plasmid DNA released from 
polylactic-polyglycolic particles. Results also indicated that repair 
fibroblasts (rat osteotomy model) in vivo will take up and express plasmid 
DNA released from block co-polymers of polylactic-polyglycolic particles. 
FIG. 7 demonstrates that repair fibroblasts (rat osteotomy model) in vivo 
will take up and express pGAM2 plasmid DNA following release from 
polylactic-polyglycolic particles. As shown in FIG. 7, expression of 
plasmid-encoded PTH1-34 is associated with significant new bone formation 
in the osteotomy gap. 
Taken together, these studies show that the gene activated matrix 
technology does not depend on a collagenous matrix for success. Therefore, 
the technology is broad enough that it can be combined with both 
biological and synthetic matrices. 
10. EXAMPLE: TRANSFER OF GENES TO REGENERATING TENDON AND TO REGENERATING 
CRUCIATE LIGAMENT IN VIVO 
There is a clinical need to stimulate scar formation during the repair of 
Achilles' tendon and ligaments (shoulder and knee) in order to enhance the 
mechanical competence of the injured tissue. A model system has been 
developed in which segmental defects in the Achilles' tendon is created 
and a novel biomaterial, small intestinal submucosa or SIS, is used as a 
tendon implant/molecular delivery agent. In the present example, the 
ability to deliver and express marker gene constructs into regenerating 
tendon tissue using the SIS graft is demonstrated. 
10.1. MATERIALS AND METHODS 
Segmental defects in Achilles tendon have been created and a preparation of 
SIS has been used as a tendon implant/molecular delivery system. Plasmid 
(pSVogal, Promega) stock solutions were prepared according to standard 
protocols (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual 
Cold Spring Harbor Laboratory Press). SIS graft material was prepared from 
a segment of jejunum of adult pigs (Badylak et al., 1989, J. Surg. Res. 
47:74-80). At harvest, mesenteric tissues were removed, the segment was 
inverted, and the mucosa and superficial submucosa were removed by a 
mechanical abrasion technique. After returning the segment to its original 
orientation, the serosa and muscle layers were rinsed, sterilized by 
treatment with dilute peracetic acid, and stored at 4.degree. C. until 
use. 
Mongrel dogs (all studies) were anesthetized, intubated, placed in 
right-lateral recumbency upon a heating pad, and maintained with inhalant 
anesthesia. A lateral incision from the musculotendinous junction to the 
plantar fascia was used to expose the Achilles, tendon. A double thickness 
sheet of SIS was wrapped around a central portion of the tendon, both ends 
were sutured, a 1.5 cm segment of the tendon was removed through a lateral 
opening in the graft material, and the graft and surgical site were 
closed. The leg was immobilized for 6 weeks and then used freely for 6 
weeks. Graft tissues were harvested at time points indicated below, fixed 
in Bouins solution, and embedded in paraffin. Tissue sections (8 .mu.m) 
were cut and used for immunohistochemistry. 
10.2. RESULTS 
In an initial study, SIS material alone (SIS-alone graft) engrafted and 
promoted the regeneration of Achilles, tendon following the creation of a 
segmental defect in mongrel dogs as long as 6 months post surgery. The 
remodeling process involved the rapid formation of granulation tissue and 
eventual degradation of the graft. Scar tissue did not form, and evidence 
of immune-mediated rejection was not observed. 
In a second study, SIS was soaked in a plasmid DNA solution (SIS+plasmid 
graft) and subsequently implanted as an Achilles' tendon graft (n=2 dogs) 
or a cruciate ligament graft (n=2 dogs) in normal mongrel dogs. A 
pSV.beta.gal plasmid that employs simian virus 40 regulatory sequences to 
drive .beta.-galactosidase (.beta.-gal) activity was detectable by 
immuno-histochemistry using a specific antibody in 4/4 mammalian hosts. As 
a negative control, .beta.-gal activity was not detected in the unoperated 
Achilles, tendon and cruciate ligament of these mammalian hosts. It 
appeared, therefore, that SIS facilitated the uptake and subsequent 
expression of plasmid DNA by neotendon cells in both tendon and ligament. 
A third study was designed to evaluate the time course of .beta.-gal 
transgene expression. SIS+plasmid grafts were implanted for 3, 6, 9, and 
12 weeks (n=2 dogs per time point) and transgene expression was assayed by 
immunohistochemistry. Cross-sections (8 .mu.m) of Bouins fixed, paraffin 
embedded tissue were cut and mounted on Probeon Plus slides (Fisher). 
Immunohisto-chemistry was performed according to the protocol provided 
with the Histostain-SP kit (Zymed). In brief, slides were incubated with a 
well characterized anti-.beta.-galactosidase antibody (12:00 dilution, 5 
Prime.fwdarw.3 Prime), washed in PBS, incubated with a biotinylated second 
antibody, washed, stained with the enzyme conjugate plus a 
substrate-chromogen mixture, and then counterstained with hematoxylin and 
eosin. 
Bacterial .beta.-gal activity was detected in tendons that received the 
SIS+plasmid graft (8/8 mammalian hosts). Although not rigorously 
quantitative, transgene expression appeared to peak at 9-12 weeks. 
Bacterial .beta.-gal gene expression was not detected in 35 mammalian 
hosts that received SIS-alone grafts. 
11. EXAMPLE: ADENOVIRAL GENE TRANSFER INTO REGENERATING BONE IN VIVO 
An alternative method to achieve in vivo gene transfer into regenerating 
tissue is to utilize an adenovirus-mediated transfer event. Successful 
adenoviral gene transfer of a marker gene construct into bone repair cells 
in the rat osteotomy model has been achieved. 
11.1. MATERIALS AND METHODS 
Adenoviral vector pAd. CMVlacZ, is an example of a replication-defective 
adenoviral vector which can replicate in permissive cells 
(Stratford-Perricaudet et al., 1992, J. Clin. Invest. 90:626-630). In this 
particular vector the early enhancer/promoter of the cytomegalovirus (CMV) 
is used to drive transcription of lacZ with an SV40 polyadenylation 
sequence cloned downstream from the reporter gene (Davidson et al., 1993, 
Nature Genetics 3:219-223). 
pAd.RSV4 has essentially the same backbone as pAdCMVlacZ, however the CMV 
promoter and the single Bg1II cloning site has been replaced in a 
cassette-like fashion with a Bg1II fragment that consists of an RSV 
promoter, a multiple cloning site, and a poly(A.sup.+) site. The greater 
flexibility of this vector is contemplated to be useful in subcloning 
osteotropic genes, such as the hPTH1-34 cDNA fragment, for use in further 
studies. 
An Ultra Fiber.TM. implant was soaked for 6 minutes in a solution of AdCMV 
lacZ virus (10.sup.10 -10.sup.11 plaque forming units or PFU/ml) and then 
implanted into the osteotomy site. The defect was allowed to heal for 3 
weeks, during which time the progress of the wound healing response was 
monitored by weekly radiographic examination. By three weeks, it was 
estimated that 40% of the defect was filled with callus tissue. The 
mammalian host was sacrificed and tissues were fixed in Bouins fixation 
and then demineralized for 7 days using standard formic acid solutions. 
11.2. RESULTS 
The results obtained conclusively demonstrated expression of the marker 
gene product in chondrocyte-like cells of the osteotomy gap (FIG. 6). The 
nuclear-targeted signal has also been observed in pre-osteoblasts. 
12. EXAMPLE: TRANSFER OF GENES TO SKELETAL MUSCLE 
There is a clinical need to stimulate scar formation during the repair of 
soft tissues besides Achilles' tendon and ligaments (shoulder and knee) in 
order to enhance the mechanical competence of the injured tissue. A model 
system has been developed in which incisions in adult rat skeletal muscle 
are made and a suture preparation coated with a preparation of sustained 
release PLGA particles and plasmid DNA is used as a skeletal muscle/gene 
delivery device. To demonstrate the feasibility of the coating 
compositions and methods of the invention, a surgical suture was coated 
with marker DNA (encoding human placental alkaline phosphatase) and used 
to suture rat muscle tissue. The experiment demonstrates successful 
transfer and expression of DNA in the tissue repaired with the coated 
suture. 
12.1 MATERIALS AND METHODS 
12.1.1 PREATION OF DNA-PLGA COATING COMPOSITION 
To 1.5 mL of a PLGA/chloroform solution (3% (w/v) 50/50 polylactic 
polyglycolic acid PLGA co-polymer, ave. MW 90,000, inherent viscosity 
1.07) was added 0.2 mL of a solution containing marker DNA encoding human 
placental alkaline phosphatase (1 mg DNA, 0.5 mM Tris-EDTA, 0.5 mM EDTA, 
pH 7.3). The solution was emulsified by vortexing for 2 minutes followed 
by sonicating for 30 seconds at about 0.degree. C. using a microtip 
probe-type sonicator at 55 Watts output. This process yielded an emulsion 
that looked very milky. 
12.1.2 COATING A SURGICAL SUTURE 
A hole was pierced in a piece of Teflon-coated foil (Norton Performance 
Plastic Corp., Akron, Ohio) using a 22-gauge needle. On the hole was 
placed a drop (about 60 .mu.L) of the DNA-PLGA emulsion. A 70 cm length of 
3-0 chromic suture (Ethicon) was drawn through the hole to coat the 
suture. As the suture passed through the hole it became coated with a thin 
(ca. 30 .mu.m-thick), uniform coating of the coating composition. The 
suture was allowed to air dry for about 3 minutes, and the coating process 
repeated 15 times, allowing each coat to air dry. The coated suture was 
examined by electron microscopy (150X) and the suture was found to be 
coated with a uniform coating of DNA-PLGA. Furthermore, the coating 
remained intact even after passing the suture through tissue multiple 
times. 
12.1.3 REPAIRING SKELETAL MUSCLE WITH THE COATED SUTURE 
The suture prepared above was sewn into the skeletal muscle tissue of two 
normal adult rats with satisfactory surgical results. The suture exhibited 
good tie-down properties. One week later, muscle plus suture was 
dissected, snap frozen in liquid nitrogen and ground into a powder. The 
powder was incubated in 200 .mu.L lysis buffer, exposed to three 
freeze-thaw cycles and clarified. The clear liquid was assayed for 
alkaline phosphatase activity using standard methods after incubation at 
65.degree. C. 
12.2 RESULTS 
The results indicated that rat skeletal muscle sewn with coated sutures and 
retrieved after one week exhibited alkaline phosphatase activity, 
signifying that the marker alkaline phosphatase gene was expressed in the 
muscle tissue. Control retrievals showed no significant alkaline 
phosphatase activity. These data demonstrate that emulsions can be used to 
effectively coat sutures and deliver genes to proliferating repair cells 
in vivo. 
13. EXAMPLE: TRANSFER OF GENES TO BLOOD VESSEL 
There is a clinical need to prevent excessive fibrosis (restenosis), as, 
for example, may occur during blood vessel repair following angioplasty. 
This might be accomplished, for example, by delivery of genes that code 
for lysyl oxidase inhibitors, or by transfer of genes that code for 
certain TGF-.beta.s. There is, in addition, a clinical need to regulate 
angiogenesis, as, for example, in vascular insufficiency disorders, where 
the goal would be to stimulate new vessel formation in order to prevent 
tissue hypoxia and cell death. A model system has been developed in which 
repair cells in large blood vessels in rabbit are transfected with a 
preparation of sustained release PLGA particles and plasmid DNA. Repair 
cells are present because these rabbit blood vessels harbor a foam cell 
lesion that mimics clinical atherosclerosis in humans. The present example 
demonstrates the ability to deliver and express marker gene constructs 
into large blood vessel repair cells. 
13.1. MATERIALS AND METHODS 
New Zealand white rabbits of either sex, weighing 3.1 to 3.5 kg, were used 
for this study. Rabbits were anesthetized using Ketamine (35/mg/Kg) and 
Xylazine (5 mg/kg) given intramuscularly, and maintenance anesthesia was 
achieved with intravenous ketamine (8 mg/kg) administered via a marginal 
vein. Approximately 2 cm Segments of both iliac arteries between the 
descending aortic bifurcation and inguinal ligament were isolated, tied 
off proximally, and all small branches of this arterial segments were 
ligated. Local thrombus were prevented by the ear-marginal vein 
administration of heparin (100 mg). Via an iliac arteriotomy, a balloon 
angioplasty catheter (2.0 mm balloon) was introduced into iliac arteric 
segments and balloon was dilated for 1-minute at 8 atm pressure. 
Following balloon dilatation, the angioplasty catheter was removed, 20 mg 
of heparin was injected intra-arterially to prevent distal thrombosis. 
Both ends of iliac artery were tightened with 10.0 silk, the 5 mg/ml 
DNA-Nanoparticle suspension was infused in each iliac artery over 3 
minutes at 0.5 atm. The wound was sutured. Rabbits were sacrificed 2 weeks 
after the balloon angioplasty and nanoparticle delivery. Through a 
vertical lower abdominal incision, both iliac arteries were isolated. A 2 
cm segment of iliac artery was excised bilaterally. Carotid arteries from 
rabbit was taken as a control sample. The tissue was preserved in liquid 
nitrogen for alkaline phosphatase assay. 
13.2. RESULTS 
The results of the phosphatase expression assays indicated that a 
nanoparticle plus DNA formulation was capable of delivering nucleic acids 
to repair cells in the iliac arterics of adult rabbits injured with a 
ballon catheter. Both the right and left iliac arterics were positive for 
phosphatase activity after exposure to nanoparticle plus DNA formulations. 
No phosphatase activity was detected in the control aorta. These positive 
results indicate upon exposure to a gene activated matrix repair cells in 
large blood vessels can take up and express nucleic acid molecules. 
The present invention is not to be limited in scope by the exemplified 
embodiments which are intended as illustrations of single aspects of the 
invention, and any clones, DNA or amino acid sequences which are 
functionally equivalent are within the scope of the invention. Indeed, 
various modifications of the invention in addition to those skilled in the 
art from the foregoing description and accompanying drawings. Such 
modifications are intended to fall within the scope of the appended 
claims. 
It is also to be understood that all base pair sizes given for nucleotides 
are approximate and are used for purposes of description.