1. Field of Invention
This invention relates generally to the identification of a novel gene, Wit 3.0, and its use in wound healing. More particularly, the invention relates to methods and compositions of treatment including, but not limited to, adult skin wound healing, soft tissue wound healing and oral mucosa wound healing.
Specifically, the present invention identifies and describes Wit 3.0, a gene, differentially expressed in oral mucosa tissues undergoing wound healing.
The present invention also provides for methods of administering Wit 3.0 to incisional and/or excisional soft tissue wounds. Delivery of Wit 3.0 is in antisense form, or alternatively, in sense form.
2. Description of the Related Art
By definition, a wound disrupts the normal epithelial barrier, which is the first line of defense against invading microorganisms. Wound healing typically results after surgery or other injuries to the tissue and involves a complex set of cellular and molecular events to restore the structural integrity of the damaged tissue. Following injury or surgical incision, there are three main stages of wound repair. The initial phase occurs during the first 24 to 48 hours after the event and is characterized by blood coagulation, inflammation and initial wound closure. The middle phase occurs during the first week following the event, and is characterized by cell proliferation and formation of granulation tissue. Granulation tissue is described as small, red, grain-like prominences that form on raw surfaces such as that of wounds or ulcers. Granulations are processes of healing. This middle phase is dependent on new blood vessel formation, or angiogenesis. The last phase, occurring several weeks into the healing and repair process, is characterized by connective tissue synthesis and remodeling.
Fibroblasts in resting tissue are quiescent, immobile cells engaged in minimal biosynthetic activities. After wounding, these cell proliferative and migrate into the wound region where they synthesize and contract a new connective tissue matrix, including collagen, which resembles smooth muscle. Once healing is complete, the wound fibroblasts again become quiescent and regress through apoptosis.
Migration of fibroblasts to the injury site followed by fibroblast proliferation is stimulated by various growth factors (i.e., epidermal growth factor-EGF, fibroblast growth factor-FGF, transforming growth factor, TGF-beta). These factors also stimulate the synthesis and secretion of extracellular matrix components such as collagen and proteoglycans. At the same time, establishment of new blood vessels (angiogenesis) is occurring and stimulated by fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF).
As healing progresses there is a decrease in the number of active fibroblasts, an increase in extracellular matrix components such as collagen and a decrease in concentration of blood vessels. During this process, the region of the scar is immobilized and collagen fibers are deposited in a near-random fashion.
For example, healing of a clean, uninfected incision where the edges have been approximated with sutures begins when the edges fill with clotted blood. The blood clot at the surface becomes dehydrated forming a scab and within a day the epidermis thickens. A few days later, granulation tissue begins to occupy the incision space and fills it in a couple of days. In the event of larger incisions or wounds, there is an increase loss of cells and tissue at the injury site and wound contraction is mediated by myofibroblasts.
Therefore, the failure of these cellular and molecular events produces abnormal scarring. Examples of abnormal scarring include keloid formation, which is an accumulation of larger than normal amounts of collagen at the wound site creating a protruding scar; proud flesh, which is an abnormal increase in the amount of granulation tissue that blocks reepithelialization; contracture formation, which is immobilized tissue resulting in undesirable fixed, rigid scars that can limit normal range of motion; and fibrosis, which is abnormal connective tissue resulting from myofibroblast activity in the extended wound.
Other factors also undermine normal wound healing repair mechanisms. In particular, systemic disease, medications, and behavioral factors such as smoking and diet can impede the normal wound repair response. For example, diabetes and peripheral vascular disease impair the formation of healthy granulation tissue such as collagen. Medications such as immunosuppressants can inhibit the inflammatory response and delay wound healing. For example, corticosteroids are immunosuppressants that have dual effects: inhibiting lymphocyte function and inhibiting the synthesis of structural skin proteins, such as collagen.
It has been widely recognized that fetal skin and oral mucosa leave minimal scarring. The wound created in these tissues commonly exhibits the rapid initial wound closure likely due to the active approximation of the wound margin within the initial phase, or 24-48-hour period. In contrast, adult skin wounds do not close as rapidly as fetal skin and oral mucosa. It has also been postulated that the adult skin wound lacks initial wound closure mechanisms and tends to create excess tissue contraction and/or fibrosis formation in the later healing stages. Active approximation, similar to that of fetal skin and oral mucosa, in the adult can be achieved by placement of sutures. For example, well-sutured wounds have about 70% of the strength of normal skin. Although, minimal scarring has been observed in fetal skin and oral mucosa, to date, the molecular mechanisms involved in the fetal skin and oral mucosa wound closure have not been elucidated.
One-third of the elderly are currently edentulous (without teeth) in either one or both jaws. Formed as a result of wound healing, edentulous mucosa is the portion of the oral mucosa that covers the site where the tooth has been removed. The wound healing process during tooth extraction is thought to follow a similar chronological and physiological pattern as that of typical soft-tissue wound healing as previously discussed.
To maximize wound healing, growth factors and small peptides that stimulate the proliferation and biosynthetic activity of cells in the wound have to be isolated and characterized. Existing pharmaceutical creme formulations containing several different growth factors including platelet derived growth factor (PDGF) and transforming growth factor beta (TGFβ) do not ameliorate the situation occurring in the edentulous oral mucosa because the cremes originate from more than one cell type
Another disadvantage of the existing formulations containing various growth factors is that the particular factor(s), which offer the greatest benefit in wound healing, cannot be specifically determined. Moreover, effects of multiple growth factors may be potentially adverse on distant organs. As a primary example, angiogenic growth factors enhance the proliferation of blood vessels and hence enhance wound repair. However, these same factors can also enhance neovascularization in areas where it is undesirable, such as accelerating the growth of any benign or malignant tumor.
Thus, an improved treatment is composed of an active agent, which is locally expressed at or near the region of the wound. Such an agent enhances wound healing at the local site of injury but is not deleterious to nearby normal healthy tissues.
Another improved treatment administers the active agent to minimize undesired effects on non-wound tissues. For example, a method of administration localizes and limits the active agent to restrict release of the factor to the wound site.