Recombinant keratinocytes

Details Recombinant keratinocytes Recombinant keratinocytes

1999-04-13

2001-07-31

Clark, Deborah J. R. (Department: 1633)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C536S023500, C435S320100, C435S455000

Reexamination Certificate

active

06268215

ABSTRACT:

FIELD OF THE INVENTION
The subject invention is directed generally to recombinant keratinocytes and uses thereof, and more particularly to keratinocytes having nucleic acid encoding a human &bgr;3 integrin subunit introduced therein and to the use of these recombinant keratinocytes in a method of enhancing wound healing in a graft procedure.
BACKGROUN OF THE INVENTION
Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
It is estimated that in 1992 (US), 35.2 million wounds required major therapeutic intervention (Medical Data International, Inc. 1993). Surgical incisional wounds are performed with aseptic technique, and are closed by primary intention. Most repair and heal uneventfully. Many traumatic wounds and cancer extirpations, however, must be left open to heal by secondary intention. Furthermore, chronic wounds have significant tissue necrosis and fail to heal by secondary intention. It is estimated that 5.5 million people in the US have chronic, nonhealing wounds and that their prevalence is increasing secondary to the increase in age-related diseases, the increase in Acquired-Immune Deficiency Syndrome (AIDS), and the increase of radiation wounds secondary to cancer intervention. In the US approximately 1.5-2.5 million people have venous leg ulcers; 300,000-500,000, diabetic ulcers; and 2.5-3.5 million, pressure ulcers (Callam et al. 1987; Phillips and Dover 1991; Lees and Lambert 1992; Lindholm et al. 1992). These acute and chronic open wounds require long-term care and procedures that include skin grafting and tissue flaps, debridement, frequent dressing changes and administration of pain medications. This care is costly and labor intensive. Furthermore, these wounds have a severe impact on the patients' quality of life. The chronic dermal ulcerations can cost as much as $40,000 each to heal and more disappointing is that 50% reappear within 18 months of healing. Chronic dermal ulcers are also associated with mortality. As many as 21% of patients in intermediate-care facilities with pressure ulcers die (Bergstrom et al. 1994).
Although multiple millions of dollars have been spent on the development of numerous recombinant growth factors (Abraham and Klagsbrun 1996; Heldin and Westermark 1996; Nanney and King 1996; Roberts and Sporn 1996) and organotypic skin replacements (Boyce et al. 1995) for use in open wounds over the past decade, the evidence of cost-effective benefit is meager thus far (Brown et al. 1989; Robson et al. 1992a; Robson et al. 1992b; Phillips et al. 1993).
One approach to promoting wound healing has been the use of grafts. Split-thickness skin grafts removed from one site of a patient and grafted to the wound site on the patient have been used. These split-thickness skin grafts involve costly procedures and considerable pain and suffering for the patient. Another approach in the use of grafts involves the in vitro culturing of keratinocytes removed from a burn victim. The cultured keratinocytes are then grafted back onto the patient (an autograft) (Phillips et al. 1990; Gallico et al. 1984).
Fetal foreskin keratinocytes from one person have been used for grafting onto another person (an allograft) (Phillips et al. 1990). A continuing problem with such allograft procedures is the inability to consistently maintain the keratinocytes at the wound site for the time necessary for stimulation of wound healing. This problem is due in part to the inability of keratinocytes to adhere to fibrin/fibrinogen. If any fibrin/fibrinogen is present at the wound site when the allograft is applied, the keratinocytes do not adhere. Since natural wound healing involves the formation of a fibrin clot, wounds must therefore be thoroughly cleaned of fibrin/fibrinogen (clot) before application of the allograft keratinocytes. This is not an easy or practical task.
Animal keratinocytes (particularly pig keratinocytes) have also been used for human graft procedures (a xenograft). Pig keratinocytes are generally not rejected by humans, but the inability to consistently maintain the pig keratinocytes at the wound site for the time necessary for stimulation of wound healing remains a problem. See Phillips 1998 for a review of developments in biological skin substitutes.
Wound healing occurs as a succession of overlapping states that include inflammatory cell infiltration, re-epithelialization, granulation tissue formation and extracellular matrix (ECM) remodeling. Previous in vivo studies demonstrate that keratinocyte adhesion and motility require expression of the appropriate cell surface receptors for ECM molecules (Woodley 1996; Clark et al. 1985a). Integrins compose one family of such receptors (Adams and Watt 1991). There are over 20 integrins which mediate cell-ECM or cell-cell interactions. Each integrin is a heterodimeric transmembrane protein, consisting of one &agr; and one &bgr; subunit in a noncovalent complex, which together specify specific ligand binding (Yamada et al. 1996).
In normal human skin, major integrins expressed by keratinocytes are &agr;2&bgr;1, &agr;3&bgr;1, and &agr;6&bgr;4 (Yamada et al. 1996). The &agr;2&bgr;1 integrin is a type I collagen receptor while the &agr;3&bgr;1 and &agr;6&bgr;4 integrins are laminin 5 receptors which form critical adhesions to the basement membrane. In contrast, during re-epithelialization the migrating epidermal cells up-regulate integrins that ligate provisional matrix proteins (Clark 1990) including &agr;5&bgr;1 (Juhasz et al. 1993; Larjava et al. 1993; Cavani et al. 1993; Gailit et al. 1994), &agr;v&bgr;5 (Gailit et al. 1994; Clark et al. 1996a), and &agr;v&bgr;6 (Larjava et al. 1993; Clark et al. 1996a; Haapasalmi et al. 1996).
Fibrinogen, the major provisional matrix protein present in the wound space immediately after injury (Clark 1996), is a 340,000-Da-hexamer composed of two A&agr;, two B&bgr;, and two &ggr; chains, and it circulates at approximately 3 gm/liter (Mosesson 1992). Endothelial cells and fibroblasts bind fibrinogen and fibrin through the &agr;v&bgr;3 integrin in vitro (Newman et al. 1996; Gailit et al. 1997) and invade the fibrin clot in vivo (Clark et al. 1996b; McClain et al. 1996). In contrast, cultured keratinocytes do not produce the &bgr;3 integrin subunit (Adams and Watt 1991) and therefore do not express the &agr;v&bgr;3 integrin and do not invade the fibrin-rich clot (Odland and Ross 1968).
The severity of the problem of chronic, nonhealing wounds dictates that continual efforts be made to define new and more effective compositions and/or methods for facilitating wound healing.
SUMMARY OF THE INVENTION
This need is met by the subject invention which provides a recombinant keratinocyte having nucleic acid encoding a human &bgr;3 integrin subunit introduced therein. This recombinant keratinocyte is able to express the &agr;v&bgr;3 integrin and therefore is able to adhere to fibrin/fibrinogen. Therefore, when used in a graft procedure, the recombinant keratinocyte is able to adhere to fibrin/fibrinogen present in any fibrin clot in the wound. The need for removal of all fibrin/fibrinogen from the wound site prior to applying the graft is therefore removed.
The invention thus further provides a method of enhancing wound healing in a graft procedure. The method comprises introducing nucleic acid encoding a human &bgr;3 integrin subunit into keratinocytes, and using the resulting keratinocytes in a graft procedure.


REFERENCES:
patent: 0846702A2 (1998-06-01), None
Garlick et al. Crit Rev Oral Biol Med. 7(3): 204-221, 1996.*
Horch et al. Cell Transplantation. 7(3): 309-317, May 1998.*
Ledley, F.D. Pharmaceutical Research 13: 1595-1613, Nov. 1996.*
Choate et al. Human Gene Therapy. 8(14): 1659-65, Sep. 1997.*
Hultman et al. Journal of Trauma Injury. 45(1): 25-34, Jul. 1998.*
Rennekampf et al. J. Surgical Research. 62(2): 288-295, May 1996.

Affiliated with

Also associated with

Rating

Recombinant keratinocytes does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Recombinant keratinocytes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant keratinocytes will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2495356