Treatment for wounds

TECHNICAL FIELD

[0002] This invention relates to the field of the cell biology of epidermal cells and the substratum, and enhancement of the properties of these tissues for purposes of therapy. The invention also relates to the enzyme telomerase reverse transcriptase, and its use in regulating telomere length.

BACKGROUND

[0003] The worldwide chronic skin wound market, which includes diabetic foot ulcers, venous stasis ulcers and bedsores, is estimated to bear over $6 billion annually in treatment costs. The number of patients is about 12.5 million. The largest proportion is the venous stasis market, estimated at $3 billion annually, or 3.6 million patients. Venous leg ulcers are a type of chronic wound that affects up to 1 million people in the U.S., 90% of whom are over age 50. Skin lesions also present for medical treatment following accidents that involve abrasion or burning of the dermis.

[0004] Pharmaceuticals under development for managing these conditions include compositions that promote activity of endogenous cells at the site of the wound.

[0005] The family of keratinocyte growth factors has been implicated in the process of wound healing. Beer et al. (J. Investig. Dermatol. Symp. Proc. 5:34, 2000) showed that KGF is weakly expressed in healthy human skin, but strongly upregulated in dermal fibroblasts after skin injury. Binding to a transmembrane receptor on keratinocytes induces both proliferation and migration of the cells, and protects them from toxic effects of reactive oxygen species. Soler et al. (Wound Repair Regen. 7:172, 1999) characterized KGF-2 as a potential wound healing agent. It was found to increase both proliferation and migration of keratinocytes, and promote healing of human meshed skin explanted grafts and surgical excisions.

[0006] U.S. Pat. Nos. 5,814,605 and 5,965,530 provide pharmaceutical compositions comprising keratinocyte growth factor (KGF-1), for use in reducing hair loss. U.S. Pat. No. 6,077,602 relates to the sequence of keratinocyte growth factor 2 (KGF-2) and variants with enhanced activity and stability, for use in promoting wound healing. KGF-2 is currently being evaluated in clinical trials for treating injuries and skin disorders.

[0007] Other options that have been proposed for promoting activity in cells near the wound include the following. U.S. Pat. No. 5,718,897 outlines a method of enhancing migration and proliferation of keratinocytes in wound healing, by treating the wound with collagenase and a growth factor. U.S. Pat. No. 5,997,863 outlines a method of enhancing wound healing by administering enzymes that degrade glycosaminoglycans such as heparin or chondroitin sulfate in various combinations. Inada et al. (Am. J. Pathol. 157:1875, 2000) propose to facilitate wound healing by activating the transglutaminase-1 gene. Jaakkola et al. (Gene Ther. 7:1640, 2000) used adenovirus to deliver the gene for growth factor inducible element named “FiRE” into wound margin keratinocytes. U.S. Pat. No. 6,001,805 provides a method of enhancing wound healing by stimulating fibroblast and keratinocyte growth in vivo using amphipathic peptides. U.S. Pat. No. 6,191,110 outlines a method of enhancing wound healing by stimulating fibroblast and keratinocyte in vivo using amphipathic peptides of a particular sequence.

[0008] Other compositions for promoting wound healing including isolated cells and cell matrices derived from the subject being treated or a third-party donor, and adapted to provide protection of the wound while healing takes place.

[0009] U.S. Pat. No. 5,980,888 relates to a biomaterial designed for treating skin wounds, in which keratinocytes are attached to microcarrier beads of 50-500 microns in diameter. International Patent Publication WO 97/08295 outlines a reconstituted skin, comprising a dermal matrix inoculated with epithelial cells or their progenitors. U.S. Pat. No. 5,861,153 outlines a skin equivalent, comprising a support, isolated keratinocytes, and Langerhans' cells that have been activated by culturing with keratinocytes or growth factors. U.S. Pat. No. 5,580,781 reports a method for treating a skin defect by applying epidermal tissue comprising cultured outer root sheath cells. U.S. Pat. No. 6,110,208 outlines an artificial human skin comprising a support comprising a microperforated membrane upon which keratinocytes have been seeded, and an underlying tissue upon which fibroblasts have been seeded.

[0010] Genetically modified epithelial cells have been investigated in several contexts. U.S. Pat. Nos. 4,868,116, 4,980,286, and 5,698,436 relate to the introduction and expression of foreign genetic material in epithelial cells. International Patent Publication WO 97/23602 outlines techniques for obtaining human skin cell lines that have been immortalized with the SF40 large T antigen, or the E6/E7 gene of HPV16.

[0011] In 1998, Organogenesis received FDA marketing clearance for its full-thickness artificial skin product, Apligraf®, for treating venous stasis wounds. Like human skin, the product has two primary layers, an outer epidermal layer made of living human keratinocytes, the most common cell type of the human epidermis, and an inner dermal layer consisting of living human fibroblasts, the most common cell type in the human dermis. The human keratinocytes and fibroblasts used in its manufacture are derived from donor tissue. Apligraf® is currently approved for treating venous leg ulcers and diabetic foot ulcers.

[0012] The considerable complexity of the wound healing process is reviewed in Science magazine (P. Martin, Science 276:75, 1997). The article takes the view that normal adult wound repair is less like patching and more like regeneration. In view of the pervasive presence of skin lesions in our aging population, there is a compelling need for new modalities in wound healing.

SUMMARY

[0013] This disclosure provides materials and methods for treating wounds. Some aspects of the invention relate to agents that activate degenerative epithelial cells to restore normal mobility, resist apoptosis, and increase their proliferative capacity. The agents increase telomerase activity in epithelial cells and other cells present near a wound site, promoting the cells to move to the site and restore an epithelial layer. Other aspects of the invention relate to compositions comprising epithelial cells in which telomerase activity has been increased, useful as grafts in the treatment of wounds.

[0014] One embodiment of the invention is a pharmaceutical composition comprising a vector encoding telomerase reverse transcriptase (TERT), or other agent that increases telomerase activity or expression, formulated for administration to a wound site or an epithelial surface, such as the skin. The agent may be provided in a suitable excipient, such as a cream or gel, which may contain a constituent that enhances penetration or resistance to proteases, or otherwise enhances or prolongs efficiency. The composition may cause transient TERT expression in cells at the wound site if it is an adenovirus or lipid vector, or permanent TERT expression in cells and their progeny if it is a retrovirus vector. Some of the many effects possible are that epithelial cells treated with the composition express certain levels of telomerase activity (as measured in a TRAP assay), the ability to migrate on a solid surface at a substantial rate, or secretion of factors or matrix materials that promote wound closing.

[0015] Another embodiment of the invention is a pharmaceutical composition comprising telomerized epithelial cells or fibroblasts. The composition may further comprise a microparticle or matrix to enhance administration to a wound site or an epithelial surface, such as the skin, and may be further accompanied by a matrix or dressing for attaching the cells to a treatment site. In certain circumstances, the telomerized cells in the composition may express certain levels of telomerase activity, or the ability to migrate on a solid surface at a substantial rate.

[0016] Other embodiments of the invention relate to treating a wound or an epithelial cell surface, using a pharmaceutical composition of this invention. Exemplary are compositions containing a vector encoding telomerase reverse transcriptase (TERT), or compositions containing telomerized epithelial cells. Included are methods in which an agent is applied that causes increased expression of TERT in cells at the wound site. Subsequently, the treatment site can be monitored for effect of the composition, such as closing of the wound or reepithelialization of the surface. Administering the composition may have a number of beneficial effects, such as enhancing wound closure compared with an untreated wound, increasing TERT activity or expression in any restorative cell type present in the wound.

[0017] Another embodiment of the invention is a method of increasing migration of an epithelial cell, comprising causing increased telomerase activity in the epithelial cell (for example, by causing increased expression of TERT in the cell). The cell may subsequently be monitored for the effect of treatment, such as telomerase activity, or the ability to migrate on a solid surface.

[0018] Another embodiment of the invention is a method for screening a compound for its ability to affect cell migration, epithelialization, or wound healing, either in vitro or in vivo. For example, the compound can be contacted with telomerized epithelial cells in culture, and the effect on migration can be determined. Alternatively, the compound can be administered to an epithelial surface comprising telomerized cells on a living subject, and the effect on the treated cells can be determined.

[0019] The pharmaceutical methods and treatment compositions can be used for any therapeutically desirable purpose, including the treatment of any epithelial surface for wounds or any other perceived imperfection. The invention is particularly suitable for treating acute lesions, such as a traumatic lesion, burn, or surgical incision; and chronic lesions, such as a chronic venous ulcer, diabetic ulcer, or compression ulcer.

[0020] Other aspects of the invention will be apparent from the description that follows.

DRAWINGS

[0021]
FIG. 1 is a map of the retroviral vector that was used to transduce keratinocytes for expression of telomerase reverse transcriptase (TERT). The human TERT encoding sequence and a puromycin drug selection gene (puro) is driven by a constitutive viral LTR promoter.

[0022]
FIG. 2 shows that TERT expression increases replicative capacity of primary human keratinocytes. Culture of adult keratinocytes (HEKa18, HEKa2) and neonatal keratinocytes (HEKn9, HEKn4) were transduced with control or TERT expression retroviral vectors, drug selected, and then serially passaged as shown.

[0023]
FIG. 3 shows that transduction of keratinocytes with the TERT retrovirus causes TERT expression, increased telomerase activity, and lengthening of telomeres. Panel (a) shows quantitation of hTERT transcripts determined by RT-PCR. Panel (b) shows telomerase activity in cell lysate, as detected by TRAP assay. The H1299 tumor cell line is a positive control. Panel (c) shows telomere terminal restriction fragment lengths of human TERT transduced keratinocytes, and vector control (BABE).

[0024]
FIG. 4 shows that transduced keratinocytes have normal expression of cell cycle regulation proteins and c-myc.

[0025]
FIG. 5 shows that growth of transduced keratinocytes is dependent on epidermal growth factor (EGF), and sensitive to phorbol ester (TPA), characteristic of normal growth control (i.e., a non-malignant phenotype).

[0026]
FIG. 6 shows the behaviour of keratinocytes in a wound healing model. Keratinocytes were grown to near confluence, and then a 1 mm streak was cleared to determine keratinocyte migration over the next 4 days.

[0027] Panel (a) shows results of the HEKn9 neonatal keratinocyte line transduced early in culture with the human TERT retrovirus vector, or with vector control (BABE). TERT expressing keratinocytes taken to 152 population doublings retained migration characteristics of very young cells (PD8), which is at least 3-fold higher than the migration rate usually observed in keratinocytes reaching their doubling limit (PD41).

[0028] Panel (b) shows results of old HEKn9 cells (PD41) transduced with adenovirus vector for transient expression of hTERT, or with vector control (AdGFP). Short-term induction of telomerase activity in these cells restored their ability to close the wound.

[0029]
FIG. 7 shows the rate of wound closure following transduction of late-passage HEKn9 keratinocytes for increased expression of TERT (or vector control). Either long-term TERT expression (from retrovirus transduction) or transient expression from adenovirus transduction) caused a comparable acceleration in wound healing over the 4-day period.

[0030]
FIG. 8 is from an experiment in which TNF-α induced apoptosis of keratinocytes was measured by Annexin V staining. HEKa2 keratinocytes treated with vector control (open bars) were ˜20% susceptible to apoptotic cell death, which increased in the presence of TNF-α. However, telomerized keratinocytes (stippled bars) showed lower levels of apoptosis, and were resistant to the effects of TNF-α.

[0031]
FIG. 9 shows that TERT also protects keratinocytes against UV irradiation induced apoptosis. For the top graph, cells were transfected with an adenovirus expressing hTERT for 3 days, irradiated for 24 h, then stained with Annexin V. hTERT stabilized the cells against apoptosis to control levels after UV irradiation up to 10 mJ cm−1. For the bottom graph, the cells were allowed to proliferate for 5 days between hTERT transfection and UV irradiation. The protective effect of hTERT is still present, suggesting that resistance to apoptosis may ensue from increased telomere length.

[0032]
FIG. 10 shows results from an experiment in which migration of keratinocytes was uncoupled from cell proliferation. Neonatal keratinocytes from the HEKn9 line (“H9”) proliferated after they were transduced to express telomerase (AdhTERT), or with vector control (AdGFP). In both cases, mitomycin c (MC) inhibited the proliferation by over 2-fold.

[0033]
FIG. 11 shows the rate of wound closure in the presence of mitomycin c (10 μg/mL).

[0034]
FIG. 12 shows the behaviour of keratinocytes in the wound healing model in the presence of mitomycin c. Telomerization of keratinocytes still increased the rate of wound closure by >3-fold, even though proliferation of the cells was inhibited by mitomycin c. This indicates that the enhanced wound closing induced by telomerase expression involves more rapid migration of the epithelial cells, independent from proliferative capacity of the cells.

[0035]
FIG. 13 shows reconstitution of telomerase activity in rabbit fibroblasts. Cultured fibroblasts were transduced with AdhTERT for 24 h, and then analyzed 48 h later for TRAP activity. Expression of the TERT gene reconstitutes telomerase activity in a dose-dependent manner.

[0036]
FIG. 14 shows hTERT gene transfer into rabbit skin tissues cultured ex vivo. An adenovirus vector encoding hTERT was injected intradermally and the tissues harvested 3 days later. Frozen tissues sections were stained with anti-hTERT antibody (top panel) and co-localized with nuclear staining using DAPI (bottom panel).

[0037]
FIG. 15 shows paraffin sections from ischemic rabbit ear wounds treated with control vector (left) or adenovirus hTERT vector. The sections show increased formation of granulation tissue in the aged rabbit ear wounds treated with AdhTERT but not in the control.

[0038]
FIG. 16 quantitates the granulation tissue in aged rabbit ischemic wounds. The granulation tissue cross-sectional area (A) and distance migrated (B) was quantitated and expressed as mean values ±SEM. There was 3.9-fold increase in granulation tissue area (Panel A) and 2.2-fold increase in migration distance (Panel B) in the group treated with the AdhTERT vector, but not the control (p<0.01).

[0039]
FIG. 17 shows AdhTERT reconstitution of telomerase activity in cultured rhesus monkey fibroblasts treated to express hTERT, as measured by TRAP assay.

[0040]
FIG. 18 shows efficient hTERT gene transfer into monkey skin. The tissue was obtained from aged rhesus monkey monkeys, and injected intradermally with buffer control (Top Panel), or AdhTERT (Bottom Panel). The panels show antibody staining for hTERT expression, co-localized with nuclear staining using DAPI. The results show that the vector caused hTERT protein expression in the dermal region.

[0041] No TRAP activity was detectable in AdhTERT transduced tissues, presumably due to low efficiency of gene transfer or expression.

[0042]
FIG. 19 shows wound closure in aged rhesus monkeys treated with AdhTERT (▪) or control vector (&Circlesolid;).

[0043]
FIG. 20 shows sections of normal human skin punches cultured ex vivo. The epidermal layer migrated along the cut edge of the punches with increasing time in culture.

[0044]
FIG. 21 (Top Panel) shows migration of epidermal cells in human skin punches cultured in different media. The Bottom Panel shows the pattern of epidermal migration for 4 normal human skin tissues over a period of 7 days. Epidermal migration rate was relatively consistent among punches obtained from the same donor.

[0045]
FIG. 22 shows expression of adenoviral delivery of hTERT to human skin punches. AdhTERT was injected into normal (left) or wound derived (right) skin punches. The cells were then stained with antibody for hTERT (upper panels), co-localized with propidium iodide (lower panels).

[0046]
FIG. 23 shows that transient hTERT expression substantially enhances epidermal migration in human skin. The Top Panel provides results from a skin sample taken from a 78 year old donor. The epidermis of untreated skin punches or punches treated with AdLacZ (negative control) stopped migrating by 3 days. In contrast, the punch treated with AdhTERT migrated for 5 days to over twice the distance.

[0047] The Bottom Panel provides results of normal skin tissue, and skin taken near a chronic wound in the same donor (GTS 1388, age 39). Epidermal migration was slower in the wound. AdhTERT enhanced migration of the wound tissues by almost 3-fold, but had no effect on the normal tissue. The effect is greater than would be expected based on the number of cells detectably expressing hTERT, indicating that the transfected cells are recruiting activity in the surrounding epithelium.

DETAILED DESCRIPTION

[0048] The healing of an adult skin wound is a complex process, requiring collaboration between different cells and tissues. The phases of healing involve proliferation, migration, matrix synthesis, and contraction of the collaborating cells. Compositions that advance these processes may provide considerable improvement to the therapeutic modalities available.

[0049] It has now been discovered that increasing telomerase activity has a variety of effects that enhance the wound-healing potential of cells near the site of the wound. Replication is enhanced, and the cells become less susceptible to triggers of apoptosis. A surprising finding made in the course of this work is that telomerase expression also substantially enhances mobility of old cells surrounding the wound—allowing them to close the wound more rapidly. This is of considerable interest, because reepithelializing open areas of the wound creates a sterile barrier, and enhances healing of the subdermal tissues.

[0050] The enzyme telomerase is known to be generally involved in maintaining telomere length and forestalling replicative senescence in dividing cells. Most normal human somatic cells possess low or undetectable levels of telomerase, and their telomeres shorten with each cell division, ultimately leading to replicative senescence.

[0051] Kang et al. (Cell Growth Differ. 9:85, 1998) found that normal human oral keratinocytes (but not fibroblasts) have levels of telomerase measurable by telomeric repeat amplification protocol (TRAP) that diminished as the cells were passaged. Harle-Bachor et al. (Proc. Natl. Acad. Sci. USA 93:6476, 1996) dissected human skin taken during surgery, and tested for telomerase levels. They found that dermal fibroblasts were telomerase negative, but the epidermis had detectable telomerase activity, attributable to proliferative basal cells, which may act to promote regeneration of the epidermis. Fujimoto et al. (Oral. Oncol. 37:132, 2001) measured telomerase expression in oral keratinocytes and squamous cell carcinomas. Campisi et al. (J. Invest. Dermatol. 3: 1, 1998) and Mendez et al. (J. Vasc. Surg. 28:876, 1998) reported that loss of telomeres, proliferative capacity, and function are associated with skin aging and chronic wounds.

[0052] Artificially increasing the expression of telomerase can prevent the onset of senescence in some normal cells, increasing replicative capacity without causing malignant transformation (Bodnar et al., Science 279:349,1998; Yang et al., J. Biol. Chem. 274:26141, 1999; Morales et al., Nature Genet. 21:115, 1999). Ectopic expression of telomerase has been found to immortalize skin fibroblasts and microvascular endothelial cells, while maintaining growth control and differentiated function (Jiang et al., Nature Genet. 21:111, 1999). Farwell et al. (Am. J. Pathol. 156:1537, 2000) determined genetic and epigenetic changes in epithelial cells immortalized by telomerase. Yang et al. (Nat. Biotechnol. 19:219, 2001) determined the effect of telomerase on human microvasculature in vivo. Funk et al. (Exp. Cell Res. 258:270, 2000) found that telomerase expression restores dermal integrity to in vitro aged fibroblasts in a reconstituted skin model.

[0053] However, before the filing of the present disclosure with the Patent Office, previous reports of epithelial cells with increased telomerase expression have taught against the invention claimed in this application. It has been reported that telomerase expression is insufficient to immortalize keratinocytes. Loss of cell cycle control was believed to be a second requirement for immortalization—specifically, inactivation of the pRb/p16INK4a pathway (Dickson et al., Mol. Cell. Biol. 20:1436, 2000; and Kiyono et al. Nature 396:84, 1998).

[0054] In spite of those discouraging reports, the experiments detailed below were conducted to determine what effect increased telomerase activity in keratinocytes would have on phenotypic features of the cells. Ectopic telomerase expression by itself was found to be sufficient for primary keratinocytes to bypass senescence and extend their life span—even in the absence of Rb/p16INK4a cell cycle control disruption. Normal levels of c-myc protooncogene expression, and normal growth and differentiation are maintained (Example 2, below). Furthermore, keratinocyte cultures established from adult donors and subsequently telomerized were shown to lose their susceptibility to apoptosis-inducing agents (Example 4).

[0055] A significant aspect of this discovery in the context of wound healing is that upon telomerization, epithelial cells from older adults acquire considerably improved capacity to mobilize and move into open areas of a wound. As shown in FIG. 6 (Example 3), keratinocytes transfected to express telomerase reverse transcriptase close a cleared 1 mm streak in tissue culture within 3 days—an improvement of at least 3-fold, compared with the vector control. The experiment described in Example 5 demonstrates that the increased mobilization is not simply due to increased proliferation rate: if the cells are treated with mitomycin c so as to block proliferation, wound closure still remains considerably enhanced.

[0056] Another remarkable finding during the course of this investigation is the ability of telomerized cells to recruit activity of other cells to promote wound closure. FIGS. 15 and 16 (Example 6) show that inducing telomerase activity at the site of a wound in animal models causes substantial increase in granulation tissue formed, expediting the healing process. FIG. 23 (Example 8) shows that telomerase preferentially affects senescent cells near the wound, causing them to revert to a younger phenotype, as illustrated by increased migration over the wound surface. The extent of improvement found in these experiments goes beyond what might be predicted from the number of cells actually expressing telomerase. The implication is that the telomerized cells secrete factors or otherwise influence neighboring cells to participate in healing.

[0057] The description that follows illustrates how this discovery can be implemented in clinical therapy in a variety of embodiments. Polynucleotide vectors and other agents can be applied to increase telomerase expression in cells around the site of a wound, thereby initiating or enhancing reepithelialization and closure of the wound over underlying tissues. Alternatively or in addition, the wound can be treated with a preparation of telomerized cells to overlay or repopulate the open area of a wound. These strategies can be implemented as effective treatments on their own, and can also be used as effective adjuncts to other wound-closing therapies.

General Techniques

[0058] For further elaboration of general techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in cell and molecular biology, tissue culture, and veterinary and human medicine.

[0059] Methods in molecular genetics and genetic engineering are described generally in the current editions of Molecular Cloning: A Laboratory Manual, (Sambrook et al., Cold Spring Harbor); Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.); and Current Protocols in Molecular Biology (F. M. Ausubel et al. eds., Wiley & Sons). Cell biology, protein chemistry, and antibody techniques can be found in Current Protocols in Protein Science (J. E. Colligan et al. eds., Wiley & Sons); Current Protocols in Cell Biology (J. S. Bonifacino et al., Wiley & Sons) and Current protocols in Immunology (J. E. Colligan et al. eds., Wiley & Sons.). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, and Clontech.

[0060] Cell culture methods are described generally in the current edition of Culture of Animal Cells: A Manual of Basic Technique (R. I. Freshney ed., Wiley & Sons); Culture of Epithelial Cells (R. I. Freshney ed., Wiley & Sons), General Techniques of Cell Culture (M. A. Harrison & I. F. Rae, Cambridge Univ. Press).

[0061] Topical publications include Molecular Biology of the Skin: The Keratinocyte (M. Darmon & M. Blumenberg, eds., Academic Press), Wound Closure Biomaterials and Devices (Chu et al. eds., CRC Press), and Biomembranes Part V: Cellular and Subcellular Transport: Epithelial Cells (S. Fleischer & B. Fleischer eds., Meth. Enzymol. vol. 191).

Cell Isolation

[0062] Skin cells and epithelial cells of various types can be isolated from tissue samples taken from humans and other species to validate the effectiveness of agents proposed for increasing telomerase levels, and to prepare some of the telomerized cell compositions of this invention.

[0063] Primary cultures of keratinocytes (skin epithelial cells) are readily obtained by culturing skin cells that have been separated by dissection and/or enzymatic digestion from a corresponding sample of epithelium, such as split-thickness explants of human skin. The cells can be passaged in serum-free medium, and form confluent, stratified cultures.

[0064] In one method, a layer of feeder cells is prepared form the 3T3 line of human fibroblasts (ATCC Accession No. CRL-1658). The feeders are grown in 3T3 medium at 37° C. to ˜50% confluence, treated with mitomycin c (1-10 μg/mL) for 12 h, and then seeded at 2.5×104 cells/cm in keratinocyte growth medium (KGM: DMEM/F12 1:3, 10% fetal calf serum, 4 mM L-glutamine, 100 U/mL penicillin & streptomycin, 0.4 μg/mL hydrocortisone, cholera endotoxin (1×10−10 M), transferrin (5 μg/mL), liothyronine (2×10−11 M), adenine (1.8×10−4 M), insulin (5 μg/mL) and EGF (10 μg/mL). A skin sample is submerged briefly in alcohol 3 times, dried, and trimmed to remove hypodermis so only the epidermis and relatively dense dermis remain. The sample is then cut into 2-3 mm thin strips, and covered with medium containing dispase at 2 mg/mL overnight at 4° C., or for 2-4 h at 37° C. The epidermis is then peeled away from the dermis using two sterile hypodermic needles, and placed into 5 mL 0.05% trypsin solution with shaking for 1 min. Fifteen mL DMEM containing 10% FCS is added to inactivate the trypsin, and pieces of the upper epidermal layer is removed by passing through sterile gauze. The flow-through single-cell suspension is then centrifuged at 300 g for 5 min, resuspended in KGM, and plated on to the feeder layers at 2-5×104 viable cells cm−2, or onto a collagen-IV coated flask.

[0065] Other methods for culturing keratinocytes are described by Rheinwald and Green (Cell 6:331, 1976), Flaxman et al. (Br. J. Dermatol. 92:305, 1975), Price et al. (J. Natl. Cancer Inst. 70:853, 1983), Wilke et al. (J. Natl. Cancer Inst. 80:1299, 1988), Germain et al. (Burns 19:99, 1993); and reviewed by Daniels et al. (Burns 22:35, 1996) and Barlow et al. (Methods Mol. Biol. 75:117, 1997). U.S. Pat. No. 5,712,163 provides chemically defined culture media for culturing epithelial cells, containing nutrients, insulin or IGF, transferrin or Fe2+, T3 or thyroxin, an ethanolamine, and calcium above 1.0 mM. Depending on the source and the culture method, doubling times can be achieved of up to 33 hours, and between 20 and 50 population doublings. Telomerase activity in the cultured epithelial cells can then be increased as described in the following section. U.S. Pat. No. 4,016,036 provides a process for serially culturing keratinocytes on a layer of inactivated fibroblast feeder cells. As an alternative, the cells can be grown on a porous analog of the extracellular matrix that supports the cells in vivo, such as collagen (Orgill et al., J. Biomed. Mater. Res. 15:39, 1998).

[0066] As an alternative, useful cell populations can be obtained by providing a population of stem cells, and then permitting or causing the cells to proliferate or differentiate into the desired phenotype. Li et al. (Proc. Natl. Acad. Sci. USA 31:3902, 1998) isolated and characterized candidate human keratinocyte stem cells. U.S. Pat. No. 6,200,806 (Thomson) and U.S. Pat. No. 6,090,622 (Gearhart et al.), and International Patent Publication WO 99/20741 (Geron Corporation) provide compositions of human pluripotent stem cells.

[0067] Tani et al. (Proc. Nati. Acad. Sci. USA 97:10960, 2000) provide enrichment methods for keratinocyte stem cells based on cell surface phenotype. Jones et al. (Cell 73:713, 1993) and International Patent Publication WO 99/47644 report enrichment of human keratinocyte stem cells to a high degree of purity using cell-surface integrins. Pellegrini et al. (Med. Biol. Eng. Comput. 36:778, 1998) provide cultivation conditions for human keratinocyte stem cells. Bata-Csorgo et al. (J. Clin. Invest. 95:317, 1995) report kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture.

[0068] Differentiation into a phenotype characteristic of certain types of epithelial cells can be determined according to characteristic morphology and cell-surface markers, such as cytokeratins (K1, K4, K10), integrins (integrin β1, α6β4 integrin), and the receptor for keratinocyte growth factor. Stem cells differentiated to the desired phenotype can then be treated to increase the level of telomerase activity. Alternatively, the stem cell can be genetically altered to increase telomerase activity in cell progeny, and then differentiated into an epithelial cell with appropriate characteristics.

[0069] The compositions and techniques of this invention are generally applicable to different types of cells at the site of a wound, including but not limited to epithelial cells such as keratinocytes, and the underlying substrata. Reference to keratinocytes in the following description serves as a model for other types of cells, and is not meant to limit the practice of the invention except where explicitly required. Cells suitable for treatment in accordance with this invention include epithelial cells of the dermis, and of the internal mucosa. Clinical aspects of this invention can be performed on human patients, and veterinary subjects such as pets, livestock, other mammals, avians, and other vertebrates, as appropriate.

[0070] Other cells of interest in the practice of this invention can be studied in situ or isolated according to any suitable technique. For example, isolation and culture of human fibroblasts is described inter alia by Houck, Sharma & Hayflick, Proc. Soc. Exp. Biol. Med. 137:331, 1971; and in U.S. Pat. Nos. 5,460,959 and 6,093,393. Fibroblasts can be recognized by their characteristic stellate or spindle shape, ability to form collagen, or ability to respond to fibroblast growth factors (FGF). Gupta et al. (Exp. Cell Res. 230:244, 1997) and Cha et al. (Yonsei Med. J. 37:186, 1996) describe techniques for isolation and culture of human dermal microvascular endothelial cells. Isolation, characterization, and culture of mucosal epithelial cells are described by Pool-Zobel et al., Environ. Mol. Mutagen. 24:23, 1994; and in International

Increasing Telomerase Activity

[0071] Increasing telomerase activity in cells according to this invention can be accomplished by any effective mechanism, including but not limited to the following:

[0072] genetically altering the cell with a nucleic acid having an encoding region for telomerase reverse transcriptase (TERT);

[0073] artificially placing TERT protein or telomerase holoenzyme into the cell;

[0074] increasing TERT expression from the endogenous gene;

[0075] increasing the activity of endogenous TERT by applying an activating small molecule drug or other compound;

[0076] altering expression, availability, or activity of some other component involved in telomerase biology (such as telomerase RNA component or a telomere-associated protein), thereby effectively increasing telomerase activity; or

[0077] any combination of these effects.

[0078] A convenient method to increase telomerase activity is to genetically alter the cells so that they express TERT, which is the limiting component of telomerase enzyme expression in most cells. A TERT gene can be cotransfected with a gene for the telomerase RNA component, or a TERT can be selected that is compatible with the RNA component already expressed by the cell. A cell is referred to in this disclosure as “telomerized”, if it has been genetically altered with a recombinant polynucleotide to increase functional telomerase activity, either on a transient or permanent basis.

[0079] The polynucleotide and amino acid sequence of human TERT is provided in SEQ. ID NOs:1 & 2. See also Nakamura et al., Science 277:955, 199; and U.S. Pat. Nos. 6,166,178 and 6,261,836, which describe the use of TERT to increase replicative capacity of various cell types. Vectors used to express human TERT typically encode at least 10, 30, or 100 consecutive amino acids in SEQ. ID NO:2, or a protein sequence that is at least 70% or 90% identical to a fragment of SEQ. ID NO:2, and having telomerase reverse transcriptase activity. The encoding sequence typically encodes at least 25, 100, or 300 consecutive nucleotides in SEQ. ID NO:1, or a nucleotide sequence 70% or 90% identical to a fragment of SEQ. ID NO:1, or hybridizes to such a sequence under stringent conditions.

[0080] When TERT is referred to in this description, it is understood to mean a polypeptide comprising a TERT sequence from any species, with or without alterations (such as insertions, mutations and deletions) with respect to the native sequence—so long as the gene product has telomerase catalytic activity when associated with telomerase RNA component, as measured by TRAP assay, described below. Mouse TERT sequence is provided in International Patent Publication WO 99/27113. Other publications with telomerase-related sequences include International Patent Publication WO 98/21343 (Amgen); WO 98/37181 (Whitehead); WO 98/07838A1 (Mitsubishi); WO 99/01560 (Cambia), and U.S. Pat. No. 5,583,016 (Geron Corp.). U.S. Pat. Nos. 5,968,506 and 6,261,556 (Geron Corp.) describes purified mammalian telomerase and methods for obtaining it.

[0081] Expression vectors embodied in this invention are polynucleotides that have an encoding region, which upon expression in a target cell, is able to confer on that cell an increase in telomerase activity. Typically, vectors with a TERT encoding sequence will further comprise a heterologous transcription control element that will promote transcription in the intended undifferentiated or differentiated cell line. Sequences that can drive expression of the TERT coding region include viral LTRs, enhancers, and viral promoters (such as MPSV, SV40, MoLV, CMV, MSCV, HSV TK), eukaryotic promoters (such as β-actin, ubiquitin, elongation factors exemplified by Ef1α, ubiquitin, and PGK) or combinations thereof (for example, the CMV enhancer combined with the actin promoter).

[0082] A TERT expression cassette can be delivered into the cell genome using a suitable vector system, such as a retrovirus or adenovirus. Transfection and expression of telomerase in human cells is described in Bodnar et al., Science 279:349, 1998 and Jiang et al., Nat. Genet. 21:111, 1999. For causing TERT expression on a permanent basis (for example, to create telomerized cells for administration), the pBABE retroviral vector shown in FIG. 1 is exemplary. For causing TERT expression on a transient basis (for example, for rejuvinating cells already present at a wound site), the AdhTERT adenoviral vector detailed in Example 4 is exemplary.

[0083] As an alternative, the replicative capacity of the cell line can be enhanced without integrating a TERT gene into the genome. For example, TERT can be transiently expressed using a suitable expression system such as adenovirus, or by introducing TERT protein (or the telomerase holoenzyme) directly into the cell. The TERT will be diluted out as the cell divides, but extension of telomeres in the parent cell should increase replicative capacity of the cell line by several doublings. Other suitable vectors include nucleic acid-lipid compositions effective for causing expression of the encoded protein, such as DNA lipofectin or lipofectamine complexes, neutral or anionic liposomes (U.S. Pat. Nos. 5,753,258, 5,756,122, 5,981,501), cationic lipid complexes (U.S. Pat. Nos. 6,008,202, 6,020,202 and 6,071,533), or combinations with amphipathic lipids (WO 00/59474).

[0084] Another alternative is to upregulate TERT expression from the endogenous gene by upregulating expression of trans-activating transcriptional regulators. The TERT promoter contains a number of regulator recognition sequences, such as c-myc, SP1, SRY, HNF-3β, HNF-5, TFIID-MBP, E2F and c-myb. See International Patent Publication WO 00/46355.

[0085] Another alternative is to deliver to the cell an enzyme capable of conferring telomerase activity. For example, telomerase can be purified by affinity techniques from cells that express the holoenzyme (U.S. Pat. No. 6,261,556). Telomerase reverse transcriptase (or an enzymatically active fragment) can be combined with telomerase RNA component (U.S. Pat. No. 5,837,857) either in solution or by cotranslation in a manner that permits reassembly into a telomerase holoenzyme. The active enzyme is then provided in a form that permits it to be translocated across the cell membrane (U.S. Pat. No. 5,059,532; WO 97/04748).

[0086] A further alternative is not to increase TERT expression, but enhance the effective activity of telomerase already present in the cell. This is effective in cells that have an endogenous level of TERT expression, such as in bone marrow progenitor cells and gonadal tissue. For example, TRF1 and TRF2 are proteins that bind to telomere repeats and regulate access of telomerase (Smogorzewska et al., Mol. Cell Biol. 20:1659, 2000). Decreasing expression of such factors may enhance the ability of telomerase to increase telomere length, thereby increasing replicative capacity of the cell. Furthermore, the presence of phosphatase inhibitors or protein kinase activators has been reported to increase telomerase activity (Li et al., J. Biol. Chem. 272:16729, 1998; Bodnar et al., Exp. Cell Res. 228:58, 1996).

Determining Telomerase Activity and the Effect on Cell Behavior

[0087] Evidence of increased telomerase expression can be obtained by a variety of techniques, including but not limited to determining gene transcript levels (for example, by Northern or RT-PCR analysis), protein expression (for example, by immunocytochemistry), or telomerase activity (for example, by primer extension assay). Extended lifespan or replicative capacity of the treated cells, while often desirable, need not be positively demonstrated for the invention to be put into practice, except where explicitly required.

[0088] Telomerase activity can be determined by TRAP assay (Kim et al., Science 266:2011, 1997; Weinrich et al., Nature Genetics 17:498, 1997), or other suitable technique (e.g., U.S. Pat. No. 5,741,677). Desirable levels of telomerase activity are at least 1, 4, 10, or 20 TPG units, calculated as described in Example 2. Evaluation of TERT expression by RT-PCR or immunoassay can be done by standard methods, using the sequences disclosed in U.S. Pat. No. 6,166,178. Absent of evidence to the contrary, it can be assumed that elevated levels of TERT transcript or protein corresponding to telomerase reverse transcriptase is an indication that the activity of telomerase in the cell is also elevated. The following assay kits are available commercially for research purposes: TRAPeze® XL Telomerase Detection Kit (Cat. s7707; Intergen Co., Purchase N.Y.); TeloTAGGG® Telomerase PCR ELISApIus (Cat. 2,013,89; Roche Diagnostics, Indianapolis Ind.); and LightCycler TeloTAGGG® human TERT quantification kit (Cat. 3,012,344).

[0089] Migration of isolated epithelial cells can be determined by plating or culturing in a monolayer, creating an adjacent free space on the substrate, and periodically observing cells moving into the free space. The migration occurs even in the absence of chemotactic factors, although the response of the cells to such factors may be of interest. The assay can also include a replication inhibitor such as mitomycin c, to decouple migration from cell replication. In a preferred method (Example 3), keratinocytes are grown as a monolayer on a standard tissue culture surface (such as a T25 flask) in regular medium until ˜80-90% confluent. A transverse area is then cleared by scraping, and migration of the cells into the cleared area is observed as a function of time. Depending on other features of the cell, migration of telomerized epithelial cells can be 1, 2, 5, or 10 cell diameters per day; or 2, 3, or 5-fold higher than cells of the same type that are untreated or treated with a control vector.

[0090] Effectiveness of compositions of this invention in closing or reepithelializing a wound can be ascertained in a suitable model. Since hTERT affects telomerase activity in non-human primates and other mammals, preclinical development is well suited to animal testing. A number of established animal models are available. Jimenez et al. (J. Surg. Res. 81:238, 1999) measured the effect of KGF-2 in linear incisions made in dorsal skin of rats. Cribbs et al. (J. Burn Care Rehabil. 19:95, 1998) tested the wound healing effect of heparin-binding EGF-like growth factor in an animal burn model. Leivo et al. (Br. J. Dermatol. 143:991, 2000) measured reepithelialization rate and protein expression in a human suction-induced wound model.

[0091] Human skin can also be transplanted onto the nude mouse for evaluating wound healing in a superficial excisional full-thickness wound. See for example Rossio-Pasquier et al., Arch. Dermatol. Res. 291:591, 1999. Epidermal wound healing can also be characterized using human skin specimens in an organ culture model. Moll et al. (J. Invest. Dermatol. 111:251, 1998) found that dissociated autologous keratinocytes promoted reepithelialization of 3 mm diameter defects made in excised skin specimens.

[0092] Repopulation of human keratinocytes and fibroblasts can be tested in a spontaneous cell sorting model. See Funk et al., Exp. Cell Res. 258:270, 2000; and Wang et al., J. Invest. Dermatol. 114:674, 2000. Two-piece silicon chambers (Renner, Germany) are surgically implanted onto the backs of SCID mice to provide an aseptic wound bed resting on the muscle fascia. Dermal fibroblasts and keratinocytes are harvested from culture and resuspended in serum-free medium. Human skin reconstitutions are initiated by placing a slurry of 6×106 keratinocytes and 6-8×106 fibroblasts (isolated as already described, or obtained from an established cell line such as BJ fibroblasts). After one week, the upper chambers are removed to allow aeration of the skin surface. The skin can then be tested for blister resistance or examined microscopically.

[0093] A full-thickness human skin xenograft model can be set up using skin samples from tissue bank or surgical discards from hospitals. The samples are trimmed of subcutaneous fat tissue and cut into pieces of 1-2 cm2. SCID mice are anesthetized using isofluorane, and 0.1 mL buprinex is administered s.c. (0.1 ml) behind the nape of neck as analgesic. A full thickness skin bed matching the size of the skin graft is created on the shaved dorsal region of the animal where there is a larger surface area and better vascular supply. One or two grafts are sutured in place using 4-0 Dermalon™ (Sherwood Davis & Geck) or 6-0 Vicryl™ (Ethicon). Any bleeding is stopped by applying gelfoam™. Petroleum jelly and telfa pad is applied, and the area bandaged using elastikon™ and conform™. The bandage and the sutures are removed 14 days later, with one change of bandage at 7 days. Scabbing ensues, and the grafts can be tested after the scabs come off, usually between 4-12 weeks.

[0094] The skin structure of the xenografts is monitored by immunohistochemistry using antibodies for human skin associated markers such as involucrin (NeoMarkers), associated with upper layers of the stratum corneum and the epidermis; collagen IV (Sigma), associated with the basal portion of the epidermis; and collagen I (Southern Biotech), associated with the dermal component. These antibodies are human-specific, and do not cross-react with murine skin. In general, the xenografts are positive for all three markers with some variability. The level of murine invasion can be determined using antibodies against human vs. mouse MHC Class I antigen. The amount of mouse cell invasion is variable from graft to graft, and increases with time post-surgery.

[0095] To monitor wound healing in the xenograft model, a 3 or 4 mm wound is created in the center of the skin xenograft using a sterile biopsy punch. Bleeding can be stopped using hemostatic sponges, and an occlusive bandage is placed on top of the wound for 2 days. Immediately before bandaging and every other day after bandage removal, the size of the wound is traced using an extra fine Sharpie® pen onto a clear, sterile Hybridwell™ strip until the wound is completely closed. Most of the wounds achieve complete closure by about 2 weeks. The size of the wound is quantified with respect to time by scanning each strip into ImageQuant™ or Photoshop™ 5.03, and performing area integration of the wound outlines with Openlab™ 2.1 or lmageQuant™. Using the best curve fit function, time to 50% and 75% wound closure is determined.

[0096] One way to determine the effect of increased telomerase expression is to deliver AdhTERT or control virus to the biopsy wound by direct intra-dermal injection, topical application, or both. For example, 1×107 to 5×108 particles are resuspended in 50 μL viral dilution buffer (saline +10% glycerol), and 10-15 μL aliquots are injected into 4 different sites i.d. using a tuberculin syringe with a 29 gauge needle. Alternatively, the virus is resuspended in 20 μL and directly applied to the wound bed. After allowing 30 minutes absorption and diffusion, the wound is bandaged using Opsite™ IV (Smith & Nephew) for 2-3 days. The kinetics of wound healing is then monitored as already described. The skin xenografts are harvested at different times following wounding for analysis of skin associated markers and telomerase expression.

[0097] Systems for testing telomerase activating agents and telomerized cells in tissue culture and animal models are illustrated below in Examples 2-8

Use of Telomerizina Agents and Telomerized Cell Preparations

[0098] The techniques and compositions provided in this disclosure can be used for a variety of desirable purposes. Such purposes include research or investigational work related to the behavior of epithelial cells or cells expressing telomerase. Of particular interest is clinical use in human or veterinary medicine, such as for the treatment of wounds or enhancement of properties of the dermis wherever desired.

[0099] Compositions for clinical use according to this invention include two categories: agents that can be used to increase telomerase activity in cells already present at or around an area of the epithelium in need of treatment; and compositions containing cells with increased telomerase activity. In general, such compositions are effective in treating a wound or otherwise enhancing properties of an epithelial surface in the body when applied individually, but they may also be used in combination where the benefits of both are desired.

[0100] Agents that Increase Telomerase Activity

[0101] Agents of this invention designed to increase telomerase activity or expression include vectors encoding TERT, agents that increase transcription of the endogenous TERT gene, and agents that affect the TERT gene product, transactivators or telomerase associated proteins in a manner that increases telomerase activity in cells near the wound that is being treated.

[0102] Compositions of this invention can be formulated for treating wounds of the skin or dermis, with or without involvement of the substratum and the underlying tissues. Compositions of this invention can also be formulated for treating wounds of other epidermal surfaces, including mucosal surfaces such as the bronchus, mouth, nose, esophagus, stomach, or intestine. Unless specifically required otherwise, the techniques and compositions of this embodiment are generally applicable to humans and other vertebrates.

[0103] Suitable TERT vectors include viral vectors, naked DNA, and DNA-liposome complexes, in which the TERT encoding region is operatively linked to transcription and translation elements active in the target cell. These vectors may include a constitutive promoter (such as the CMV or EF1α promoter), or a tissue-specific promoter (such as promoters for cytokeratins or integrins expressed in epithelial cells, or the receptor for keratinocyte growth factor).

[0104] When this disclosure refers to administration of an agent “to a wound site”, what is meant is that the agent is placed at, in, or around the wound in one or more locations, such that cells at the site of administration are caused to express increased telomerase activity or increased expression of TERT. The type of cells that may be affected include epithelial cells, keratinocytes, microvascular cells, and other cells subjacent to the affected surface or exposed during wounding. It is understood that most agents of this invention administered with a view to increasing telomerase activity in a particular cell type, such as an epithelial cell, will inevitably also affect other cell types in the vicinity. Evidence of telomerase expression or clinical benefit in the general area of the wound is a desired object, and it is not necessary to understand the effect on a particular cell type at the treatment site in order to practice the invention.

[0105] The therapeutic composition will contain an amount of the agent effective for accomplishing one, two or more than two of the following effects: a) increase in the level of telomerase activity or TERT expression in epithelial cells at the treatment site; b) increase in the level of telomerase activity or TERT expression in fibroblasts or other cells at the treatment site; c) increase the mobility of epithelial cells on a solid surface (as determined in an in vitro assay); d) cause reepithelialization of a wound or epithelial surface; and e) increase the rate of wound closure or healing as determined by clinical criteria. These effects may be obtained in a single dose, or by sequential administration of two or more doses after an appropriate interval. The amount given per dose depends on the efficiency of the agent or vector chosen. For example, retroviral vectors are typically used at a titer of about 106 to 107 per mL, adjusted empirically.

[0106] General aspects of formulation and administration of pharmaceutical compounds can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton Pa.). With respect to the use of nucleic acid vectors in therapeutic applications, the reader may wish to consult The Skin and Gene Therapy (U. R. Hengge & B. Volc-Platzer eds., Springer Verlag, 2000), or Gene Therapy (Advances in Pharmacology, Vol 40) (J. T. August, J. Coyle & M. W. Anders eds., Academic Press 1997).

[0107] The agent may be administered in an excipient suitable for topical administration, or administration to a wound site. This means that the excipient will have one or more of the following three properties: a) enhanced ability to penetrate the dermis or tissues at the wound site (compared with a neutral isotonic solvent); b) enhanced ability for keeping the agent at the site long enough to enhance the effect; or c) ability to prolong activity of the agent when administered to the dermis or the wound site. Excipients that enhance penetration contain organic solvents or additives such as alcohol, oils, glycols, and emoluments, or specific carriers that cause binding to the target cell. Excipients that keep the agent at the target site include creams, gels, and semisolid compositions, or solutes that produce a semisolid or high viscosity medium once applied. Excipients that prolong longevity of the agent after administration depend on the nature of the effective agent. For example, protein or virus compositions will persist longer on the skin or at a wound site if it is prepared in an excipient that contains protease inhibitors, such as metal chelators that inhibit metalloproteinase. Similarly, bare nucleic acid compositions will persist longer in an excipient that contains nuclease inhibitors. If helpful in enhancing the shelf life, the composition may be distributed in separate components to be combined just before administration.

[0108] The agent may alternatively or in addition be administered in a device suitable for topical administration, or administration to a wound site. Typically, the device will have the characteristic of either enhancing penetration or keeping the agent at the site long enough to enhance the effect. Devices of this nature include solid matrixes made of collagen, laminin, or other biocompatible polymers, and standard dressings (such as pads or bandages) made of gauze, nylon, or various plastics. The device is typically adapted to stay in place at the site of treatment by conforming to the shape of the site, and having fasteners or positions for accommodating fasteners that allow it to be attached to the site. The product may be distributed as a combined composition, in which the device is impregnated with the agent, and designed to deliver the agent upon attachment. Alternatively, the product may be distributed as a kit, comprising the therapeutically effective agent, and a device for preparing the treatment site, or for applying the agent to the treatment site, or for covering the site during or after treatment (such as a suitable dressing).

[0109] At the option of the manufacturer or distributor, the pharmaceutical composition may be packaged with (or marketed using) a written indication for use of the product in treating wounds or the epithelium according to the invention.

[0110] Telomerized Cell Compositions

[0111] Isolated cells with increased telomerase expression or activity can be assembled into a therapeutic composition in several different forms. Generally, the composition will contain telomerized epithelial and/or fibroblast cells matched to the species and type of wound being treated: for example, keratinocytes and fibroblasts for skin lesions; mucosal epithelial cells for lesions to the gastrointestinal tract. The cells may further be engineered to express other factors that promote wound healing, such as growth factors or cytokines (e.g., KGF or FGF).

[0112] In one embodiment, telomerized epithelial cells are prepared as a suspension in a pharmaceutically compatible excipient, such as a buffer or semi-solid gel. Siedler et al. (Arch. Dermatol. 136:676, 2000) propose human fibrin glue containing keratinocytes for healing of chronic ulcers. The epithelial cells are optionally accompanied by other cells that facilitate engraftment or support the cells after engraftment, such as fibroblasts, endothelial cells, or Langerhans cells, which may or may not be telomerized.

[0113] In another embodiment, the cells are attached to a solid carrier from which they can migrate once applied to the wound. Suitable carriers include microcarriers (particles of any shape less than 1000 microns in diameter, with particles in the 100 micron range being preferred), and made of a compatible matrix such as collagen. See Voigt et al. (Tissue Eng. 5:563, 1999) and LaFrance et al. (Tissue Eng. 5:153, 1999). The large surface-to-volume ratio of the microspheres can provide a vehicle for delivering appropriate cell numbers while minimizing the amount of biomaterial to be absorbed. The composition is then applied directly to the wound cavity or ulcer, or to the region surrounding the wound from which the cells can migrate.

[0114] In another embodiment, the cells are provided in the form of a flat sheet. This may be advantageous for providing more immediate protection, or treating areas that have a paucity of proliferation-competent endogenous epithelial cells. In general, the sheet will comprise a two-dimensional arrangement of epithelial cells, supported in some manner by a porous matrix produced by other cells, or manufactured artificially using a biocompatible polymer (such as collagen, laminin, or other matrix proteins). The epithelial cells may in some cases be underlaid by a supportive layer of cells such as fibroblasts that enhance engraftment or shelf life. In accordance with this invention, cells in the composition can be either telomerized before forming into sheets, or the sheet can be preformed ex vivo (or isolated from a donor), and then telomerized using one of the vectors described earlier. If fibroblasts are contained in the composition, they may also be telomerized. The sheet is then prepared for transport, and grafted onto the wound site in the clinic.

[0115] U.S. Pat. No. 4,304,866 describes a method of producing transplantable sheets by culturing keratinocytes in a vessel and then detaching a sheet of cells from the vessel with a neutral protease such as dispase. U.S. Pat. No. 5,759,830 provides a three-dimensional fibrous scaffold containing attached cells for producing vascularized tissue in vivo. Orgill et al. (J. Biomed. Mater. Res. 39:531, 1998) outline the use of island grafts of artificial skin, comprising keratinocytes and a copolymer of collagen and chondroitin sulfate. International Patent Publication WO 99/63051 outlines a bioengineered flat sheet graft prosthesis comprising layers of processed tissue material.

[0116] When this disclosure refers to administration of a cell composition “to a wound site”, what is meant is that the composition is placed over, in, or around the wound, so as to provide coverage of at least part of the wound, or create a site from which the administered cells can migrate into the wound and promote closure or healing.

[0117] The cell compositions of this invention intended for clinical or veterinary use can be provided in an isotonic excipient, prepared under sufficiently sterile conditions for administration to the subject. They are optionally provided on a microparticle or matrix suitable for topical administration or administration to a wound site. This means that the microparticle or matrix is either adapted to adhere to the site of administration (using fasteners or dressing, if needed); or that the microparticle or matrix provides a vehicle from which the cells can migrate into the treatment site and participate in coverage of the site, reepithelialization, or healing.

[0118] Duration of the graft cells at the treatment site may be temporary or permanent, depending on the nature of the condition being treated and concurrent therapies. For permanent engraftment, it may be desirable to use compositions in which the cells are autologous or histocompatible with the patient being treated, although this is not always required. The product may be packaged as a single composition suitable for immediate use, or it may be packaged as a kit with component parts in separate containers to be admixed before administration, or for sequential administration. The kit may also contain a dressing or other substance for covering the site or improving engraftment. At the option of the manufacturer or distributor, the pharmaceutical composition may be packaged with (or marketed using) a written indication for use of the product in treating wounds or the epithelium wherever needed.

Conditions Suitable for Treatment

[0119] The techniques and compositions of this invention may be used for the treatment of wounds or other conditions of the epidermis wherever desired.

[0120] Some of the medical conditions that can be treated according to this invention are acute conditions (such as lesions suffered in trauma, burns, abrasions, surgical incisions, donor graft sites, and lesions caused by infectious agents). Other medical conditions that can be treated are chronic conditions (such as chronic venous ulcer, diabetic ulcer, compression ulcer, pressure sores, and ulcers or sores of the mucosal surface). Included are skin or epithelial surface lesions caused by a persistent inflammatory condition or infection, or by a genetic defect (such as keloid formation and coagulation abnormalities). This invention also contemplates manipulation of the skin and repair of any perceived defects in the skin surface for other purposes, such as cosmetic enhancement.

[0121] In the usual course of therapy, the treatment site is monitored for response to treatment. Desirable effects for agents that increase telomerase expression or activity include cell proliferation or migration at the treatment site, epithelialization of the surface, closure of a wound if present, or restoration of normal physiological function. Throughout this disclosure, “epithelialization” or “reepithelialization” of a treatment site means that the site acquires an increased density of epithelial cells as a result of the therapy that is applied.

[0122] Desirable effects for cell compositions include coverage of the treatment site, survival of the engrafted cells, lack of immune rejection, closure of the wound if present, or restoration of normal physiological function. The engrafted cells may participate in wound closure either by participating directly in the healing process (for example, becoming part of the healed tissue), or by covering the wound and thereby providing an environment that promotes healing by host cells.

[0123] Ultimate choice of the treatment protocol, dose, and monitoring is the responsibility of the managing clinician.

[0124] Other Uses of the Invention

[0125] Isolated cells, compositions, and mixed cell populations of this invention can also be used for any other desirable research, developmental, or therapeutic purpose. The high proliferative capacity and high mobility of telomerized epithelial cells can be maintained as the cells are passaged in culture, thereby providing a standardized reservoir of cells for further investigation. Cell cultures or matrixes can be combined with a putative therapeutic or cosmetic agent, and any alteration in cell viability, proliferation, migration, or other phenotypic feature can be correlated with efficacy of the agent. Telomerized cells can also be used in living wound models such as those described earlier, to screen the ability of other compounds to promote cell migration or the process of reepithelialization.

[0126] The examples that follow are provided by way of further illustration, and are not meant to limit the claimed invention.

EXAMPLES

Example 1

Telomerization of Keratinocytes

[0127] To determine the effect of telomerase on human keratinocytes, early passage (<PD5) cultures of both neonatal and adult keratinocytes were grown in an optimized medium and transfected with a vector encoding human telomerase reverse transcriptase (hTERT).

[0128] Human primary epidermal keratinocytes were obtained from Cascade Biologics (Portland, Oreg.). The cell lines are referred to in this disclosure according to their Cascade lot designation: HEKa18, HEKa2, HEKn9 and HEKn4 are two lines of adult keratinocytes and two lines of neonatal keratinocytes.

[0129] The cells were cultured in EpiLife™ serum-free medium plus calcium chloride at 0.06 mM and Human Keratinocyte Growth Supplement (HKGS) (Cascade Biologics, Portland, Oreg.). Cells were plated at 2-4×105 cells per T75 flask, refed every 2-3 days, and subcultured 4-7 days before high cell density was reached. PD (the number of population doublings) for every passage was calculated as log2 (number of cells at time of subculture/number of cells plated). Cumulative PD was plotted against time in culture so that replicative life span, senescence, slow growth or crisis, and immortalization could be assessed. Cells were considered to have been immortalized when the life span of a culture was greater than 50 PDs beyond that of parental cell line, and growth curves showed no sign of a decrease in proliferation rate.

[0130]
FIG. 1 is a map of the amphotrophic retroviral vector that was used to transduce cells for expression of telomerase reverse transcriptase. The hTERT encoding sequence and a puromycin drug selection gene (puro) is driven by a constitutive viral LTR promoter (Nakamura et al., Science 277:955, 1997). Control cultures were infected with an equivalent vector without hTERT. Viral titers were determined by the infection of NIH-3T3 cells with BABE-puro-hTERT or control BABE-puro vectors, and were typically 3-5×106/mL.

[0131]
FIG. 2 shows proliferation potential of control and hTERT-expressing human primary keratinocytes. Early life span cultures of two adult keratinocyte lines (HEKa18, HEKa2) and neonatal lines (HEKn9, HEKn4) were transduced with control (BABE) or hTERT expression retroviral vectors, drug selected, and then serially passaged.

[0132] Control HEKa and HEKn cultures senesced at PD 33-38 and PD51-56 respectively, as evidenced by complete cessation of cell division, senescence-associated (SA) β-galactosidase positive staining, and enlarged cellular morphology. In contrast, hTERT-transduced keratinocytes had indefinite lifespans and were negative for SA-β-galactosidase staining. Moreover, all hTERT-keratinocytes exhibited no slow phase growth or crisis stage, during which clonal populations with pRb/p16ink4a inactivation could have emerged

Example 2

Characterization of Telomerized Cells

[0133] Total RNA was isolated from keratinocytes using High Pure™ RNA Isolation Kit (Roche). 100 ng total RNA was used for real time PCR quantitation of hTERT and hTR (the telomerase RNA component) with a light cycler (Roche). TeloTAGGG™ hTERT and hTR quantitation kits (Roche) and PCR were used according to the manufacturer's protocol. Telomerase activity was assessed by the PCR-based telomeric repeat amplification protocol (TRAP) assay (Kim et al., Nucl. Acids Res. 25:13, 1997). Mean telomere restriction fragment (TRF) lengths were determined by Southern blotting (Bodnar et al., Science 279:349, 1998).

[0134]
FIG. 3 shows the effect of hTERT transduction on hTERT expression, telomerase activity and telomere dynamics in keratinocytes. Panel (a) shows quantitation of hTERT transcripts in four lines of hTERT transduced keratinocytes (transcripts per 100 ng RNA×10−6). Panel (b) shows telomerase activity in the hTERT transduced keratinocytes at various population doublings. Cell lysate equivalent to 100 cells was used for each lane. The H1299 tumor cell line is a positive control. HT=reaction mixture heat treated before PCR; IC=internal control. Panel (c) shows terminal restriction fragment lengths of keratinocytes transduced with hTERT or control vector (BABE).

[0135] Telomerase activity was quantitated using the formula

TPG
=100×[(TP−TP′)/TI]/[(R8−B)/RI]

[0136] where TP is telomerase products from test sample, TP′ is products from heat-inactivated control, TI is internal control of sample, R8 is products from quantification standard, B is buffer blank, and RI is internal control of standard. The total product generated (TPG) is defined as 0.001 amol (600 molecules) of primer TS extended for at least three telomeric repeats by telomerase in the sample. One TPG corresponds roughly to the telomerase activity in one immortal cell. Values obtained are shown in Table 1:

1TABLE 1Telomerase Activity in hTERT-Transduced KeratinocytesSampleTPG ValueHEKa18h TERT-PD235.6HEKa18h TERT-PD717.2HEKa9h TERT-PD1816HEKa9h TERT-PD9010.4H1299 (control)4.5

[0137] The transduced keratinocytes expressed relatively high levels of hTERT transcripts that increased with passage, likely reflecting enrichment of telomerase-expressing cells (Panel A). This level of expression is roughly 100-200 fold greater than that seen in tumor cell lines such as H1299 and Raji. Expression of hTR (the RNA subunit of telomerase) was steady and similar between hTERT-keratinocytes and vector controls (data not shown). hTERT-keratinocytes had high levels of telomerase activity and elongated telomeres, while control keratinocytes were telomerase negative and telomeres progressively shortened with passage (Panels B & C).

[0138] pRb phosphorylation is required for progression through the S phase. pRb activity is regulated by proteins such as CDK4, cyclin D1 and p16 (Weinberg et al., Cell 81:323, 1995). To determine whether there were perturbations in the pRb/p16 pathway in hTERT-transduced keratinocytes, expression of pRb and p16 proteins was analyzed by Western blot analysis.

[0139] Western analysis for p16 (G175-1239, PharMingen), pRb (G3-245, PharMingen), p53 (OP29, Oncogene), cyclin D1 (G124-326, PharMingen), CDK4 (DCS-35, PharMingen), c-myc (N-262, Santa Cruz Biotechnology), GADD45 (H-165, Santa Cruz Biotechnology) and TFIIB (SC-225, Santa Cruz Biotechnology) was performed as described in Wang et al. (Nature 405:755, 2000). The antibody to pRb recognizes both hyper- and hypo-phosphorylated forms of the proteins (Jiang et al., Nature Genet 21:111, 1999).

[0140]
FIG. 4 shows the expression of cell cycle regulation proteins and c-myc in hTERT-keratinocytes. (a) Vector control (BABE) and hTERT-expressing keratinocytes were maintained at either subconfluent cultures (S) or confluent cultures for 72 hours (C) and analyzed for pRb, p53, cyclin D1, CDK4, and TFIIB. (b) Vector control and hTERT-expressing keratinocytes were analyzed for p16INK4a protein levels at early and late population doublings (PDs). (c) Vector control (B) and hTERT-keratinocytes at different PDs were analyzed for c-myc and GADD45 expression. TFIIB protein was used to normalize loading in panels (b) and (c).

[0141] It was found that pRb was predominantly hyperphosphorylated in subconfluent, proliferating keratinocytes, but was hypophosphorylated when the cells were maintained at confluence (Panel A). Levels of pRb were also down-regulated at confluence. Cyclin D1 and CDK4 were expressed at similar levels in proliferating hTERT-transduced and control keratinocytes, but cyclin D1 expression was down-regulated upon growth arrest (Panel a). The amount of p16 increased in late passage keratinocytes (Panel b). In contrast to previous reports, it was found that all hTERT-keratinocytes retained stable p16INK4a protein levels even after dramatic life span extension (Panel b).

[0142] p53 plays an important role in initiation of senescence-associated growth arrest (Sedivy et al., Proc. Natl. Acad. Sci. USA 95:9078, 1998). In these experiments, it was found that p53 was normally expressed in hTERT-transduced keratinocytes in both growing and non-growing states (Panel A). Thus, neither pRb/p16INK4a nor p53 inactivation are required for immortalization of human keratinocytes by telomerase.

[0143] Wang et al. (Nature 405:755, 2000) reported that hTERT-driven cell proliferation and immortalization are associated with activation of the c-myc protooncogene. This was after long-term culture of immortalized epithelial cells that had suffered previous inactivation of the pRb/p16INK4a pathway. However, it has now been discovered that hTERT-immortalized normal keratinocytes at both early and late passages, show that c-myc and GADD45 (a downstream target of c-myc) were expressed at levels similar to that seen in control populations (FIG. 3, Panel C).

[0144] Telomerase-transduced cultures were examined under conditions known to induce arrest and differentiation of young keratinocytes: high cell density, high calcium concentrations, EGF removal, TGF-β treatment, or exposure to phorbol ester.

[0145]
FIG. 5 shows long-term retention of normal keratinocyte growth control mechanism by keratinocytes transduced with the hTERT retroviral vector (“T”, first 4 series), or vector control (“B”, next 2 series). SCC-4 is a squamous cancer cell line (positive control). Cells were plated at low density in EpiLife™ medium, either in the presence or absence of EGF; or in the presence or absence of 12-O-tetra-decanoylphorbol-13-acetate (TPA). Cells were counted 7-8 days later, and growth rate under these conditions was determined (average ±S.D of three experiments).

[0146] Under these conditions, the fractions of cycling hTERT-keratinocytes were similar to that of control cells. In contrast, the SCC-4 human squamous cell carcinoma cell line was not dependent on EGF or inhibited by phorbol ester. These results indicate that hTERT-immortalized keratinocytes retain normal c-myc expression and growth regulatory mechanisms.

Example 3

Telomerized Cells Close Wounds More Rapidly

[0147] Human keratinocyte migration and proliferation are essential for re-epithelialization of skin wounds. In this experiment, the effect of replicative senescence and hTERT-transduction in a culture model of wound closure was examined.

[0148] Keratinocytes were plated at 1×105 cells/T25 flask. Once the cells reached 80-90% confluence, the monolayer of cells was scratched in a standardized manner with a plastic apparatus to create a cell-free zone approximately 1 mm across.

[0149] Retrovirus transduction for permanent expression was effected using the hTERT/BABE vector described in Example 1. When the keratinocytes were growing in log phase, the medium was replaced with 5 mL viral supernatant in DMEM/F12 medium at a titer of 3-5×106 mL−1. After culturing overnight at 37° C. in 5%CO2/95%, the cells were washed twice in PBS, and selected for 7 days in EpiLife™ medium containing 0.5 μg/mL puromycin, and then grown in regular EpiLife™ medium.

[0150] Adenovirus transduction was effecting using a replication-deficient (E1 and E3 deleted) adenovirus, containing an expression cassette in which the hTERT encoding region is under control of CAG (CMV enhancer, chicken β-actin promoter, and the rabbit β-globin polyadenylation signal). When the keratinocytes were ˜80-90% confluent, the well was scratched to create a cell-free zone, and simultaneously transduced with the adenovirus vector at 2-10 MOI in EpiLife™ medium (1 MOI≡1 PFU≡0.7 TCID). The cells were cultured overnight at 37° C. in 5%CO2/95%, washed twice in PBS, and then grown in regular EpiLife™ medium.

[0151] In vitro re-epithelialization or wound closure was documented by photography through a 40×objective over a 1-4 day period. The width of the wound was measured at three different places in each of three replicate plates, and the rate of wound closure was calculated by linear regression of the mean wound width as a function of time.

[0152]
FIG. 6, Panel (a) shows results of transduction for TERT expression using the retroviral vector. When measured by the time required to produce a 50% wound closure (T50), it was found that young keratinocytes (PD8) closed culture wounds at a rate roughly 3-fold faster (T50=33±1.2 h) than that seen with old keratinocytes (PD41) (T50=113±5.7 h, p<0.00002). Stable hTERT expressing keratinocytes transduced at early passage, on the other hand, retained their youthful rates of wound closure (T50=32±0.8 hr, p<0.0002), even at very late passages (PD152).

[0153] To test whether telomerase could rescue age-associated deficits in wound closure in this model system, late-passage cultures of keratinocytes were wounded and then transduced with adenoviral vector for transient hTERT expression (AdhTERT). The identical adenovirus containing GFP in place of hTERT was used as a control.

[0154]
FIG. 6, Panel (b) shows the results of transduction with the adenovirus vector. Old keratinocytes were efficiently infected with adenovirus since 60-70% of cells were positive for expression 7 days after infection with AdGFP at 10 MOI and high levels of telomerase activity were seen when AdhTERT was used (data not shown). Short-term hTERT expression in late passage keratinocytes (PD42) remarkably accelerated wound healing in vitro, as shown by a near-complete wound closure on day 4 in AdhTERT-treated (T50=34±2.3 h) but not AdGFP-treated keratinocytes (T50=109±17.7 h, p<0.001). The rate of closure of the transient hTERT transduced cultures was similar to that of young cells.

[0155]
FIG. 7 shows the rate of wound closure over the 4 days following transduction for increased expression of TERT, or with a control vector. Either long-term expression (resulting from retrovirus transduction) or transient expression (resulting from adenovirus transduction) caused a comparable acceleration in wound healing over the 4-day period.

Example 4

Telomerized Cells are Resistant to Apoptosis

[0156] Cells transduced with the hTERT retrovirus were measured for their resistance to apoptotic cell death, induced by TNF-α or UV irradiation.

[0157] Apoptosis is characterized in the early stages by translocation of membrane phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane. Annexin V is a 35-36 kDa calcium-dependent binding protein with a high affinity for PS, which can be used to stain for externalized PS in early apoptosis.

[0158] For TNF-α induced apoptosis, keratinocytes were transduced with hTERT retrovirus or BABE control. The transduced cells were then cultured for 48 hours in standard keratinocyte culture medium, or medium containing TNF-α. The cells were washed in PBS containing 0.5% BSA (or 1% FBS). 5×105 cells were combined with 0.5 mL 1×Binding Buffer from the Annexin V FITC Kit. 5 μL Annexin V FITC and 10 μL propidium iodide were added, and the mixture was incubated at room temperature in the dark for 10 min. They were then measured for percentage positive cells and mean fluorescence intensity by flow cytometry.

[0159]
FIG. 8 shows the results. Adult keratinocytes treated with vector control (HEKa2 BABE PD22) were ˜20% susceptible to apoptotic cell death, which increased in the presence of TNF-α. However, only ˜10% (<2-fold less) of the telomerized keratinocytes (HEKa2 hTERT PD25) showed evidence of apoptosis, and were resistant to the effects of TNF-α.

[0160] For apoptosis induced by UV irradiation, primary adult keratinocytes were seeded in 100 mm TC dishes at 3×105 per dish, and cultured in EpiLife® medium. Cells reached about 40% confluence at 3 days, and were transduced in fresh medium containing AdhTERT at 10 MOI. AdhTERT is a replication-deficient, E1 and E3 regions deleted, adenovirus containing a cassette encoding the human telomerase gene under the control of CAG (comprising the CMV enhancer, chicken actin promoter, and a portion of 3′ untranslated region containing the polyadenylation site of rabbit globin gene). After culturing with AdhTERT for 72 h, the cells were washed twice with Ca++- and Mg++-free PBS. UV irradiation was performed for 24 h, and the cells were then stained with Annexin V.

[0161]
FIG. 9 (Top) shows the results of this experiment. Transfection with the hTERT adenovirus vector protected the keratinocytes against UV-induced irradiation at doses up to 10 mJ cm−1.

[0162]
FIG. 9 (Bottom) shows that the protective effect of hTERT is retained as the cells divide. In this experiment, cells were transfected with the AdhTERT for 3 days at 40% confluence, and then cultured under conditions that allow cell proliferation. On day 8 (when the cells were 75% confluent), they were subject to UV irradiation for 24 h, and then washed and stained the next day.

[0163] The results show that the protective effects of hTERT extend to the progeny of the cells transfected on day 8. Since adenovirus vectors provide only transient expression, the long-lasting effect may ensue from the lengthening of telomeres caused by hTERT in the parent cells.

Example 5

Enhanced Wound Closure by Telomerized Cells Does not Depend on Cell Relication

[0164] In the previous examples, telomerase expression was shown to increase replicative capacity of keratinocytes, render them less susceptible to apoptosis, and increase their capacity to re-epithelialize a wound. In this experiment, the wound healing effect was decoupled from the proliferation effect, showing that wound closure is not due simply to an increase in cell replication.

[0165] Keratinocyte cell lines were plated at 1×105 per T25 flask. Once they had grown to 80-90% confluence (˜5×105), the cell monolayer was scratched as before to create a cell-free zone. The cells were treated with mitomycin c at 10 μg/mL for 2 hours. The medium was then aspirated and replaced with fresh EpiLife™ medium, with or without Adeno-hTERT or Adeno-GFP (control), to transiently increase telomerase expression. After transducing overnight, the medium was replaced with fresh medium, and the rate of wound closure was measured for 4 days in triplicate.

[0166]
FIG. 10 shows the effect of mitomycin c (MC) on cell proliferation. The cells were trypsinized and counted on day 4 to determine the extent of proliferation since mitomycin c treatment. The HEKa18 line (“H18”) was at PD37 when plated in this experiment. This is near the full extent of its normal replicative capacity (FIG. 2). Accordingly, little further proliferation was observed, regardless of whether mitomycin c was present. The HEKn9 line (“H9”) was at PD42 when plated, which is below its full replicative capacity (FIG. 2). This cell line proliferated through several doublings when cultured in regular medium. However, mitomycin c reduced the proliferation rate by well over 50%.

[0167]
FIGS. 11 & 12 show the effect of mitomycin c (10 μg/mL) on cell migration of HEKn9 keratinocytes transduced to express hTERT (“AdT”), compared with vector control (“AdG”). The transient expression of hTERT accelerated wound closure by over 3-fold, even in the presence of mitomycin c.

[0168] A summary of the kinetics of epithelial cell migration is shown in Table 2.

2TABLE 2Kinetics of Wound ClosureT50(hours to achieve 50%Samplewound closure)HEKn9 pBABE PD833.0 ± 1.2HEKn9 pBABE PD41113.4 ± 5.7 HEKn9 PD42 + AdGFP108.9 ± 17.7HEKn9 PD42 + AdGFP + Mitomycin c189.2 ± 28.9HEKn9 pBABE/TERT PD15231.6 ± 0.8HEKn9 PD42 + AdhTERT34.3 ± 2.3HEKn9 PD42 + AdhTERT + Mitomycin c40.3 ± 2.1pBABE = retrovirus control pBABE/TERT = retroviral vector for expressing TERT AdGFP = adenoviral vector for expressing GFP (control) AdhTERT = adenoviral vector for expressing TERT

[0169] In conclusion, it has been found that hTERT-treated keratinocytes have increased replicative capacity, and are resistant to apoptosis. They retain normal growth control, as shown by dependence on epidermal growth factor (EGF) and sensitivity to phorbol ester (TPA). hTERT-treated keratinocytes do not spontaneously activate c-myc, and retain functional p53 and pRB/p16ink4a cell cycle checkpoint. Both stable and transient hTERT expression increases migration and accelerates wound healing in aging keratinocytes.

Example 6

Enhanced Wound Healing Using hTERT in the Aged Rabbit Ischemic Ear Model

[0170] In this study, it was shown that AdhTERT gene delivery induces a specific and robust enhancement of granulation tissue formation in the ischemic ear wounds of aged rabbits.

[0171] Methods

[0172] The adenovirus vector encoding hTERT under control of the CAG expression system was described in Example 4. Rabbit fibroblasts were obtained from ATCC (CRL-1414), grown in BME +10% FBS to passage 33, infected with AdhTERT or Ad-null for 24 hr at different MOI, and analyzed 48 hrs later for telomerase activity using the TRAP assay. Skin tissues were obtained from young rabbits and maintained in DMEM +10% FBS ex vivo. The tissues were injected intradermally with 2×109 viral particles and harvested 3 days later. Frozen tissue sections were analyzed for hTERT expression using anti-hTERT antibody as described below.

[0173] Ear wounds were induced in rabbits as an established clinically relevant model for wound ischemia (Ahn, S. T. & T. A. Mustoe, Ann Plast Surg 24:17, 1990; Wu et al., Am J Pathol 154:301, 1999). New Zealand white rabbits (>55 months of age) were prepared by shaving the ears and prepping with betadine solution. An incision was made to the level of bare cartilage at the base of each ear. Both ears of each rabbit were made ischemic by dissecting the rostral and central arteries, with preservation of the caudal, central and rostral veins. The incision was closed with a running 4-0 Vicryl™ suture. Three to five full thickness (6 mm) circular wounds were then made on the inner surface of the ear down to bare cartilage.

[0174] Adenoviral gene transfection was performed by delivering 2×109 viral particles of AdhTERT or Ad-null (control) per ear wound. Two thirds of total dose was injected at 4 periwound locations at 5 μL each, using a Hamilton syringe with a 30 gauge needle. One third of the dose was topically placed within the defect in 10 μL. Sterile Tegaderm™ dressing (3M Health Care, St. Paul, Minn.) was placed over each wound upon completion of the procedure. The dressings were changed as needed over the next 12 days, at which time the animals were sacrificed and the wounds harvested for histological and biochemical analysis.

[0175] Telomerase activity was measured according to standard TRAP assay procedures described earlier, as applied to frozen skin tissue homogenized in lysis buffer.

[0176] Immunohistochemical analysis of hTERT expression was performed on 6 μm frozen tissue sections fixed in 4% paraformaldehyde in PBS (pH 7), rinsed in PBS and permeabilized in PBS containing 0.1% Triton™ X-100. The sections were blocked in 5% goat serum in PBS for 30 min at room temp, drained and incubated with anti-hTERT antibody (1A4, 2.5 μg/ml) for 1 h. After washing several times in PBS, Texas-Red™ conjugated goat anti-mouse IgG (Jackson Immunolabs, Westgrove, Pa.) was added at 7.5 μg/mL for 30 min at room temp in the dark. The sections were then washed again with PBS, mounted using Vectashield™ mounting medium containing DAPI (Vector Labs), and viewed under a Nikon fluorescent microscope.

[0177] Data were collected from histological sections to determine the extent of wound re-epithelialization and new granulation tissue formation. The wound healing parameters were measured twice using a calibrated reticle from H&E-stained paraffin tissue sections by observers blinded to treatment. Analysis of all wound parameters was performed by Student's t-test and analysis of variance with post hoc analysis using Tukey's standardized range. All comparisons were made to paired wounds. Any dependent associations were analyzed using Spearman's correlation of coefficients.

[0178] Results

[0179]
FIG. 13 shows reconstitution of telomerase activity in rabbit fibroblasts, which do not express detectable endogenous telomerase. Cultured fibroblasts were transduced with AdhTERT at 0, 10, 100 or 1000 MOI for 24 h, and then analyzed 48 h later for TRAP activity. For each group, 4000 and 40,000 cell equivalents were loaded in the first and second lane, respectively. The triangle denotes lysates (40,000 cells) that were heat-inactivated prior to assay. AdhTERT but not Ad-null (the control vector) was effective in reconstituting telomerase activity in a dose-dependent fashion. Subsequent immunocytochemical analysis also showed hTERT positive cells in AdhTERT transduced rabbit fibroblast cultures.

[0180]
FIG. 14 shows hTERT gene transfer into rabbit skin tissues cultured ex vivo. AdhTERT or Ad-null was injected intradermally and the tissues harvested 3 days later. Frozen tissues sections were stained with anti-hTERT antibody (red fluorescence, top panel) and co-localized with nuclear staining using DAPI (blue fluorescence, bottom panel). hTERT protein was expressed mostly in the dermal region. However, no TRAP activity was detectable in AdhTERT transduced tissues, most likely due to the low efficiency of gene transfer/expression.

[0181] To determine if hTERT expression in rabbit skin can enhance wound healing, AdhTERT or Ad-null was administered to ischemic ear wounds of aged rabbits by both intradermal injection and topical application. Pilot experiments using young rabbits showed that AdhTERT causes hTERT expression in the dermal regions 3 days after wounding and virus administration . Analysis of the aged wounds at day 12 also showed hTERT positive dermal cells, albeit at less frequency, probably due to the transient nature of adenoviral gene expression.

[0182]
FIG. 15 shows H&E stained paraffin sections from ischemic rabbit ear wounds treated with Ad-null (left) or AdhTERT (right) and harvested on day 12. There was a dramatic increase in granulation tissue formation in the aged rabbit ear wounds treated with AdhTERT, but not in wounds treated with Ad-null. Table 3 summarizes the quantitative data.

3TABLE 3Histological analysis of aged rabbit isohemic ear woundsNoWound parametersTreatmentAd-nullAdhTERT(day 12 post-wounding)(n = 5)(n = 15)(n = 9)Granulation tissueArea (×104 μm2) 5 ± 1 7 ± 2 27 ± 6*Distance (μm)340 ± 5 445 ± 45 986 ± 152*Peak to peak distance (μm)5245 ± 1805048 ± 102 3890 ± 330*Peak height (μm)335 ± 28312 ± 27407 ± 20Epithelial tissueEpithelial gap (μm)2750 ± 8221529 ± 4681167 ± 519Epithelial height (μm)145 ± 23124 ± 14130 ± 18*p < 0.01 between Ad-null and AdhTERT

[0183]
FIG. 16 shows granulation tissue formation in aged rabbit ischemic wounds. Ischemic rabbit ear wounds were treated with Ad-null, AdhTERT or no treatment and then harvested 12 days later. The granulation tissue cross-sectional area (A) and distance migrated (B) was quantitated and expressed as mean values ±SEM. There was 3.9-fold increase in granulation tissue area and 2.2-fold increase in migration distance in the AdhTERT treated group relative to the Ad-null or no treatment group (p<0.01). However, no difference was observed in the growth or migration of the overlying epithelium.

[0184] The results show that transient expression of hTERT can specifically enhance new granulation tissue formation, which is critical in effecting wound healing. The lack of observable effect on epithelial growth or migration is most likely due to the inefficient gene delivery to the epithelium.

[0185] The hTERT effect on granulation tissue formation is quite dramatic, despite the relative inefficient gene transfer to the skin. This suggests that in addition to influencing the phenotype and/or replicative capacity of the transduced cells, hTERT expression cells may indirectly influence the phenotype of neighboring cells—for example, by elaborating trans-acting factors or altering the extra-cellular matrix environment. There was no abnormal inflammatory response in the hTERT treated wounds beyond that observed with normal wound healing, suggesting that local AdhTERT gene delivery can be used safely.

Example 7

Wound Healing in Aged Rhesus Monkey Monkeys

[0186] The ability of hTERT gene to reconstitute function in rhesus monkey cells was demonstrated by positive hTERT protein expression and telomerase activity following AdhTERT transduction of rhesus monkey fibroblasts in culture.

[0187]
FIG. 17 shows AdhTERT reconstitution of telomerase activity in culture. Rhesus monkey lung fibroblasts (NIA AG11856A) were grown in DMEM +10% FBS to population doubling 8.3, infected, transduced with AdhTERT at 0, 50, 100 or 500 MOI for 24 hrs and then analyzed 48 hrs later for TRAP activity. For each group, 1000 and 5000 cell equivalents were loaded in the first two lanes, respectively. The triangle denotes lysates (5,000 cells) that were heat-inactivated prior to assay.

[0188] The results show that monkey skin fibroblasts do not express detectable endogenous telomerase activity. The weak signals in the heat inactivated lanes are likely to be due to leakage from other adjacent lanes. Upon transduction with AdhTERT but not Ad-null, telomerase activity was reconstituted and the level of telomerase activity showed a dose related increase with the transducing viral dose. Immunocytochemical analysis also revealed hTERT positive cells in AdhTERT transduced rhesus monkey fibroblast cultures.

[0189]
FIG. 18 shows the efficiency of hTERT gene transfer into monkey skin. The tissue was obtained from aged rhesus monkeys and maintained in DEME +10% FBS ex vivo. The tissues were injected intradermally with buffer control (Top Panel), or AdhTERT (2×109 viral particles, Bottom Panel) and harvested 3 days later. The panels show antibody staining for hTERT expression, co-localized with nuclear staining using DAPI (Example 6). The results show that AdhTERT caused hTERT protein expression in the tissue, mostly in the dermal region. No TRAP activity was detectable in AdhTERT transduced tissues, presumably due to low efficiency of gene transfer or expression.

[0190] Wound healing experiments were conducted using an established model in aged rhesus monkeys (Roth et al., J Gerontol A Biol. Sci. Med. Sci. 52:B98-102, 1997). Full thickness wounds were created in female rhesus monkey monkeys (18-32 years old) anesthetized with ketamine (15 mg/kg) and diazepam (1 mg/kg). Four separate 5 mm punch biopsy wounds were made on the dorsal side of the animals. AdhTERT or Ad-null virus was applied at 1010 viral particles per wound to two wounds at the time of wounding. To measure wound closure, each monkey served as its own control. AdhTERT was used to treat 2 of the wounds on each animal, and Ad-null was administered to the other 2 wounds. Two thirds of each dose was delivered around the wound edge by 8 intra-dermal injections of 5 μL using a Hamilton syringe with a 30 gauge needle. The remaining third of the viral dose was applied topically into the wound defect (20 μL). The percentage of wound area remaining was assessed every other day. Wound tracings were performed using a single-layer plastic film placed over the biopsy site and % wound area remaining was quantified as number of pixels using NIH Image analysis software. Upon complete healing, an 8 mm punch biopsy was collected around each wound and processed for histological and biochemical analysis.

[0191] The AdhTERT vector was found to cause hTERT expression in the dermal regions 3 days after wounding and virus administration. FIG. 19 shows the results of the wound healing measurements. Each data point represents the percent wound area remaining averaged for the 2 wound receiving AdhTERT (▪) or control vector (&Circlesolid;). The effect of transient hTERT expression on wound healing in this model was inconclusive. The adenovirus vector administration did not cause abnormal inflammation, which shows that transient induction of hTERT gene expression in wounds can be done safely.

Example 8

AdhTERT Gene Delivery Promotes Epidermal Migration in Human Skin Tissues

[0192] Chronic ulcers are characterized by impaired wound healing and frequently repeated wounding at the same sites. They may be partially due to the compromised regenerative capacity of skin cells as a consequence of replicative senescence. In addition, the aberrant gene expression/phenotype often associated with the state of senescence may further exacerbate the pathology found in chronic wounds.

[0193] To extend the other findings provided in this disclosure, an assay of ex-vivo epidermal migration was developed using intact human skin tissues. The tissue was obtained from both normal donors and from donors with chronic wounds, and was used to determine the effect of hTERT gene expression on epidermal migration.

[0194] Human skin tissues from autopsy or surgical procedures were provided by Research Tissue Recovery Network (Blue Springs, Mo.) and by Dr. Spencer Brown at University of Texas Southwestern Medical Center (Dallas, Tex.) within 24 hr of isolation. Normal skin tissues were obtained from donors without any wounds or from anatomical sites distal from any affected wounds; wound tissues were obtained from sites close to or at the edges of affected acute or chronic wounds.

[0195] Upon receipt, skin tissues were trimmed of subcutaneous fat and washed 5 times using DMEM supplemented with streptomycin (10 μg/mL) and penicillin (10 units/mL). Generally, 4 or 6 mm full thickness punches were made from the skin samples using a sterile biopsy (uni-punch, Premier Medical Products, King of Prussia, Pa.). The skin punches were attached to the bottom of Petri dishes or 6-well tissue culture plates using skin closure glue Nexabond™ (Veterinary Products Laboratories, Phoenix, Ariz.), submerged in DMEM supplemented with 10% FBS and Pen/Strep, and incubated at 37° C. with 5% CO2 for up to 7 days. For each time point, 3 skin punches were harvested and fixed in 10% neutral buffered formalin for 24 h. The tissues were paraffin embedded on edge and 6 micron serial sections were generated. For each skin punch, 3 sections at different depths were stained with H&E and examined under a microscope. Photomicrographs of the sections were taken under a 2.5× object lens and the images saved as JPG files. To cover the entire tissue section, sometimes two overlapping photomicrographs were taken and assembled using Adobe PhotoShop® software.

[0196]
FIG. 20 shows H&E sections of normal human skin punches cultured ex vivo. The epidermal layer was distinguished from the dermal region in H&E stained sections due to the difference in cellularity. The epidermal layer migrated along the cut edge of the punches with increasing time in culture. Distance migrated by the epidermal keratinocytes over the cut edge of the dermis was measured on both sides using the NIH Image 1.62 software. The pixel numbers were converted into millimeters by normalizing to the original width of the punches (4 or 6 mm).

[0197]
FIG. 21 (Top Panel) shows migration of epidermal cells in different media. One was the basic fibroblast medium (DMEM plus 10% FBS) and the other was a 1:1 mixture of fibroblast medium and keratinocyte medium EpiLife™ (Cascade Biologics, Inc., Portland, Oreg.). No significant difference in the distance migrated by the epidermis was observed.

[0198]
FIG. 21 (Bottom Panel) shows the pattern of epidermal migration for 7 normal human skin tissues over a period of 7 days. 3 skin tissues were of questionable quality due to compromised shipping procedure. Epidermal migration was observed in the other 4 tissues tested. Migration of the epidermis occurred as early as day 1, and plateaued by day 3 or 5. The epidermal layer eventually reached the interface of the dermal and connective tissues and no more migration was observed. The distance migrated in certain samples decreased after 5 days, presumably due to thickening or contraction of the tissues upon long term culture. The epidermal migration rate was relatively consistent among punches obtained from the same donor.

[0199]
FIG. 22 shows expression of adenoviral delivery of hTERT to human skin punches. AdhTERT was injected into normal (left) or wound derived (right) skin punches from donor GTS 1384. Frozen tissues sections were harvested on day 3 (normal) or day 5 (wound tissue), fixed in 4% paraformaldehyde, and permeabilized in 0.1% Triton X-100™. The sections were blocked in 5% goat serum, incubated with hTERT antibody (1A4, 2.5 μg/mL) for one hour at room temp, and then stained with Texas-Red™ conjugated goat anti-mouse IgG (Jackson Immunolabs).

[0200] Upper panels show hTERT staining; lower panels show co-localization with propidium iodide. Administration by injection caused hTERT expression to be mostly localized along the injection path. Bathing with AdhTERT (108 pfu/mL for 24 h) was less efficient in transducing the dermal cells, although a few cells lining the migrating epidermis did show hTERT expression.

[0201] To assess the effect of hTERT on epidermal migration, skin punches were treated with AdhTERT by direct injection or bathing. Results were compared with punches exposed to adenovirus encoding LacZ, adenovirus control (Ad-null), or no virus. One sample of the four tested (GTS 1384, age 78, normal skin) showed significant enhancement (FIG. 23, Top Panel). The epidermis of untreated skin punches or punches treated with AdLacZ stopped migrating by 3 days. In contrast, the punch treated with AdhTERT migrated for 5 days to over twice the distance.

[0202]
FIG. 23 (Bottom Panel) shows results of normal tissue, and tissue taken from a chronic wound in the same donor (GTS 1388, age 39). Epidermal migration was slower in the wound tissues than the normal tissue, demonstrating impaired healing properties. AdhTERT enhanced migration of the wound tissues by almost 3-fold, but had no effect on the normal tissue.

[0203] These results show that hTERT preferentially affects dermal tissues (normal or pathologic) that have sub-optimal epidermal migration. hTERT transduction is not mitogenic, nor does it significantly change the phenotype of young cells. But in older cells, hTERT enables the cells to proliferate further, and causes beneficial (“youthful”) changes that result in enhanced migration and epithelializing potential. Even a few hTERT expressing cells can rescue the senescent phenotype and generate growth factors or extracellular matrix components that improve epidermal cell migration over the wound surface.

[0204] The compositions and procedures described in this disclosure can be effectively modified by routine optimization without departing from the spirit of the invention embodied in the claims that follow.

Sequence Data

[0205]

4

TABLE 4

Sequences Listed in this Disclosure

SEQ.

ID

NO:
Descriptive Annotation
Source

1

Homo sapiens
telomerase
GenBank Locus NM 003210.

reverse transcriptase (TERT)
See also Nakamura et al.,

mRNA sequence
Science 277:955, 1997; and

GenBank Locus AF015950

2

Homo sapiens
telomerase
GenBank Locus NM 0032107.

reverse transcriptase (TERT)

amino acid sequence

[0206]

5

SEQ. ID NO: 1

1
gcagcgctgc gtcctgctgc gcacgtggga agccctggcc ccggccaccc ccgcgatgcc

61
gcgcgctccc cgctgccgag ccgtgcgctc cctgctgcgc agccactacc gcgaggtgct

121
gccgctggcc acgttcgtgc ggcgcctggg gccccagggc tggcggctgg tgcagcgcgg

181
ggacccggcg gctttccgcg cgctggtggc ccagtgcctg gtgtgcgtgc cctgggacgc

241
acggccgccc cccgccgccc cctccttccg ccaggtgtcc tgcctgaagg agctggtggc

301
ccgagtgctg cagaggctgt gcgagcgcgg cgcgaagaac gtgctggcct tcggcttcgc

361
gctgctggac ggggcccgcg ggggcccccc cgaggccttc accaccagcg tgcgcagcta

421
cctgcccaac acggtgaccg acgcactgcg ggggagcggg gcgtgggggc tgctgctgcg

481
ccgcgtgggc gacgacgtgc tggttcacct gctggcacgc tgcgcgctct ttgtgctggt

541
ggctcccagc tgcgcctacc aggtgtgcgg gccgccgctg taccagctcg gcgctgccac

601
tcaggcccgg cccccgccac acgctagtgg accccgaagg cgtctgggat gcgaacgggc

661
ctggaaccat agcgtcaggg aggccggggt ccccctgggc ctgccagccc cgggtgcgag

721
gaggcgcggg ggcagtgcca gccgaagtct gccgttgccc aagaggccca ggcgtggcgc

781
tgcccctgag ccggagcgga cgcccgttgg gcaggggtcc tgggcccacc cgggcaggac

841
gcgtggaccg agtgaccgtg gtttctgtgt ggtgtcacct gccagacccg ccgaagaagc

901
cacctctttg gagggtgcgc tctctggcac gcgccactcc cacccatccg tgggccgcca

961
gcaccacgcg ggccccccat ccacatcgcg gccaccacgt ccctgggaca cgccttgtcc

1021
cccggtgtac gccgagacca agcacttcct ctactcctca ggcgacaagg agcagctgcg

1081
gccctccttc ctactcagct ctctgaggcc cagcctgact ggcgctcgga ggctcgtgga

1141
gaccatcttt ctgggttcca ggccctggat gccagggact ccccgcaggt tgccccgcct

1201
gccccagcgc tactggcaaa tgcggcccct gtttctggag ctgcttggga accacgcgca

1261
gtgcccctac ggggtgctcc tcaagacgca ctgcccgctg cgagctgcgg tcaccccagc

1321
agccggtgtc tgtgcccggg agaagcccca gggctctgtg gcggcccccg aggaggagga

1381
cacagacccc cgtcgcctgg tgcagctgct ccgccagcac agcagcccct ggcaggtgta

1441
cggcttcgtg cgggcctgcc tgcgccggct ggtgccccca ggcctctggg gctccaggca

1501
caacgaacgc cgcttcctca ggaacaccaa gaagttcatc tccctgggga agcatgccaa

1561
gctctcgctg caggagctga cgtggaagat gagcgtgcgg gactgcgctt ggctgcgcag

1621
gagcccaggg gttggctgtg ttccggccgc agagcaccgt ctgcgtgagg agatcctggc

1681
caagttcctg cactggctga tgagtgtgta cgtcgtcgag ctgctcaggt ctttctttta

1741
tgtcacggag accacgtttc aaaagaacag gctctttttc taccggaaga gtgtctggag

1801
caagttgcaa agcattggaa tcagacagca cttgaagagg gtgcagctgc gggagctgtc

1861
ggaagcagag gtcaggcagc atcgggaagc caggcccgcc ctgctgacgt ccagactccg

1921
cttcatcccc aagcctgacg ggctgcggcc gattgtgaac atggactacg tcgtgggagc

1981
cagaacgttc cgcagagaaa agagggccga gcgtctcacc tcgagggtga aggcactgtt

2041
cagcgtgctc aactacgagc gggcgcggcg ccccggcctc ctgggcgcct ctgtgctggg

2101
cctggacgat atccacaggg cctggcgcac cttcgtgctg cgtgtgcggg cccaggaccc

2161
gccgcctgag ctgtactttg tcaaggtgga tgtgacgggc gcgtacgaca ccatccccca

2221
ggacaggctc acggaggtca tcgccagcat catcaaaccc cagaacacgt actgcgtgcg

2281
tcggtatgcc gtggtccaga aggccgccca tgggcacgtc cgcaaggcct tcaagagcca

2341
cgtctctacc ttgacagacc tccagccgta catgcgacag ttcgtggctc acctgcagga

2401
gaccagcccg ctgagggatg ccgtcgtcat cgagcagagc tcctccctga atgaggccag

2461
cagtggcctc ttcgacgtct tcctacgctt catgtgccac cacgccgtgc gcatcagggg

2521
caagtcctac gtccagtgcc aggggatccc gcagggctcc atcctctcca cgctgctctg

2581
cagcctgtgc tacggcgaca tggagaacaa gctgtttgcg gggattcggc gggacgggct

2641
gctcctgcgt ttggtggatg atttcttgtt ggtgacacct cacctcaccc acgcgaaaac

2701
cttcctcagg accctggtcc gaggtgtccc tgagtatggc tgcgtggtga acttgcggaa

2761
gacagtggtg aacttccctg tagaagacga ggccctgggt ggcacggctt ttgttcagat

2821
gccggcccac ggcctattcc cctggtgcgg cctgctgctg gatacccgga ccctggaggt

2881
gcagagcgac tactccagct atgcccggac ctccatcaga gccagtctca ccttcaaccg

2941
cggcttcaag gctgggagga acatgcgtcg caaactcttt ggggtcttgc ggctgaagtg

3001
tcacagcctg tttctggatt tgcaggtgaa cagcctccag acggtgtgca ccaacatcta

3061
caagatcctc ctgctgcagg cgtacaggtt tcacgcatgt gtgctgcagc tcccatttca

3121
tcagcaagtt tggaagaacc ccacattttt cctgcgcgtc atctctgaca cggcctccct

3181
ctgctactcc atcctgaaag ccaagaacgc agggatgtcg ctgggggcca agggcgccgc

3241
cggccctctg ccctccgagg ccgtgcagtg gctgtgccac caagcattcc tgctcaagct

3301
gactcgacac cgtgtcacct acgtgccact cctggggtca ctcaggacag cccagacgca

3361
gctgagtcgg aagctcccgg ggacgacgct gactgccctg gaggccgcag ccaacccggc

3421
actgccctca gacttcaaga ccatcctgga ctgatggcca cccgcccaca gccaggccga

3481
gagcagacac cagcagccct gtcacgccgg gctctacgtc ccagggaggg aggggcggcc

3541
cacacccagg cccgcaccgc tgggagtctg aggcctgagt gagtgtttgg ccgaggcctg

3601
catgtccggc tgaaggctga gtgtccggct gaggcctgag cgagtgtcca gccaagggct

3661
gagtgtccag cacacctgcc gtcttcactt ccccacaggc tggcgctcgg ctccacccca

3721
gggccagctt ttcctcacca ggagcccggc ttccactccc cacataggaa tagtccatcc

3781
ccagattcgc cattgttcac ccctcgccct gccctccttt gccttccacc cccaccatcc

3841
aggtggagac cctgagaagg accctgggag ctctgggaat ttggagtgac caaaggtgtg

3901
ccctgtacac aggcgaggac cctgcacctg gatgggggtc cctgtgggtc aaattggggg

3961
gaggtgctgt gggagtaaaa tactgaatat atgagttttt cagttttgaa aaaaa

SEQ. ID NO: 2

MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDP

AAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFA

LLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFV

LVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPA

PGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPA

RPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYS

SGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPL

FLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQ

LLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQEL

TWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTET

TFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFI

PKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLG

LDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYC

VRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSL

NEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAG

IRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEAL

GGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRR

KLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPT

FFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVT

YVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

[0207]

Treatment for wounds

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Inventors

CPC

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Abstract

Description

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CROSS-REFERENCE TO RELATED APPLICATIONS

Provisional Applications (1)