WO1998048012A1 - Fibroblasts with a foreign gene-containing composition for treating wounds - Google Patents

Fibroblasts with a foreign gene-containing composition for treating wounds

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Publication number
WO1998048012A1
WO1998048012A1 PCT/EP1998/002038 EP9802038W WO9848012A1 WO 1998048012 A1 WO1998048012 A1 WO 1998048012A1 EP 9802038 W EP9802038 W EP 9802038W WO 9848012 A1 WO9848012 A1 WO 9848012A1
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Prior art keywords
egf
fibroblasts
gene
cells
wound
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PCT/EP1998/002038
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German (de)
French (fr)
Inventor
Roland Mertelsmann
Felicia Rosenthal
Peter Kulmburg
Björn G. STARK
Eszter Tanczos
Jürgen Kopp
Original Assignee
KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues ; Not used, see subgroups
    • C12N5/0602Vertebrate cells
    • C12N5/0652Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656Adult fibroblasts
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00Genetically modified cells
    • C12N2510/02Cells for production

Abstract

A composition for treating wounds comprises fibroblasts which contain at least one foreign gene coding for a cytokine that promotes wound healing and at least another component that promotes wound healing.

Description

Fibroblasts with a foreign gene-containing composition for

Treatment of wounds

The present invention relates to a composition for the treatment of skin damage, which are difficult to treat. In extensive burns, it can be a significant problem, restore the destroyed by damaging skin areas, especially when overwhelming parts of the skin are severely damaged. In other diseases such as tumors or chronic skin damage, it is often constitute a very significant problem, restore the damaged skin areas.

Gene transfected fibroblasts per se are already known from the prior art. In the international patent application WO 95/07105 a method for inhibiting or preventing the growth of tumor cells is described in the central nervous system of a patient, wherein the patient's immune response is stimulated by immunization with either gene-transfected tumor cells or unmodified tumor cells and gene-transfected autologous fibroblasts. The used cytokines are primarily those which stimulate the immune system. DE-A 44 06 073, which was also filed on behalf of the Hospital of the Albert-Ludwigs-University of Freiburg, relates to a general process for the production of human, clonogenic fibroblasts and a process for Gentransfizierung of fibroblasts. topical application or Gentransfizierung is not disclosed in this application specifically promote with such genes wound healing.

WO 92/15676 describes the use of gene-transfected fibroblasts for somatic gene therapy. The transfected fibroblasts are fixed in an extracellular collagen matrix and implanted under the skin. The task of this application aims aim to treat genetic defects in the human genome. In this case, a functionally active "spare" gene is introduced into a somatic cell and used to compensate for the damage caused by the defective gene. is not addressed in this reference, the treatment of wounds or

Wound healing-promoting cytokines such as TGF-α, EGF or b-FGF. In the citation especially cytokines with other biological properties are mentioned, such as GM-CSF, TNF, or EPO. The mentioned in claim 5 "fibroblast growth factor" is used as a blood vessels forming substance.

Also, various acts on keratinocytes growth factors are known. Subject of WO 90/08771 is the genetic engineering providing growth factor proteins that affect epiteliale cells. In this application, the human keratinocyte growth factor but is used as a substantially pure protein.

In US Patent 5,302,701 the development of an artificial polypeptide is described which is both cell adhesive, as has also zellwachstu sfordernde activity. This is a combination of fibronectin and fibroblast growth factor (FGF). Although U.S. Patent 5,196,196 relates to a wound dressing which comprises a carrier matrix, however, there is an essential difference from the present invention in terms of the operating principle and the active agent. In U.S. Patent purified protease nexin-I (PN-I) is used in the wound dressing. But This is not a growth factor, but an enzyme with specific properties. The protease nexin-I is a serine protease inhibitor; a member which is synthesized by human fibroblasts in culture and secreted serine protease superfamily. In the US patent, the protein is used in purified form and not in the form of a gene that is expressed by fibroblasts, into which this gene has been incorporated.

From the prior art is the use of cultured autologous keratinocytes, which are suspended in fibrin glue, known for the treatment of large-area burn wounds [Stark et al., Eur. J.Plast. Surg. (1995) 18, pp 267-271]. An attempt was also already in the pig model to use keratinocytes in wound healing transfected in situ with the aid of particle-DNA transfer [Andree et al. , Proc .Acad.Sci. USA, (1994) 91, pp 12188- 12192].

Andreatta-van Leyen et al. [J. Biomedical Materials Res., Vol. 27 (1993), pp 1201-1208] describe a wound bandage which comprises gene transfected keratinocytes, the bovine growth hormone (bGH) produce.

Known from the prior art solutions fall in part to Keratinozytenpräparationen (eg, so-called. Keratinocyte sheets) returns that are very difficult to handle and can only be used often when spatial structures have formed in the cell cultivation. There is a risk that allogeneic preparations, in particular those derived from different donors and are not characterized with viruses (HIV, HCV or yet uncharacterized viruses) are contaminated and that can be infected by the patient to be treated.

Accordingly, the present invention provides compositions for treating wounds comprising fibroblasts, the coding include at least one foreign gene for a wound-healing promoting cytokine and at least one other wound healing promoting component. In addition, the composition of the invention may comprise other ingredients which are commonly used in such compositions.

Upon further promoting wound healing component may be a so-called fibrin glue. Such fibrin glue are commercially available and are used in various fields of medicine, particularly in surgery.

In the fibrin in principle the final phase of blood coagulation is used. The fibrin glue includes fibrinogen, which is converted by thrombin to fibrin monomers. This in turn forms by end-to-end or side-to-side attachment aggregated fibrin. In addition, the fibrin glue includes in a preferred embodiment of fibronectin. At the same time, thrombin activates factor XIII, which is usually present in the fibrin sealant in sufficient quantity. The fibrin glue contains as further component a thrombin solution, which is often added together with calcium chloride as a separate component.

In addition, the fibrin sealant may also contain sufficient amounts of albumin or plasminogen.

In the further wound-healing promoting component can also be in a preferred embodiment by a fixed part, which serves as a support matrix. Such carriers for "artificial skin" are commercially available (for example Laserskin® or Biobrane®). In the solid components can preferably be a matrix of a derivative of hyaluronic acid, preferably a Hyaluronsäureester. When hyaluronic acid is a component of the body's connective tissue that is subject to an extremely high turnover rate. Thus, if the matrix is ​​composed of hyaluronic acid, it is broken down in the wound very quickly by the body's hyaluronidase. Therefore, according to the invention preferred are those derivatives of hyaluronic acid used, which are somewhat more slowly degraded in the body.

A particular advantage which can be achieved by the use of a carrier matrix is ​​that cells can be applied to the wound, which still do not form a confluent cell cluster. Although keratinocytes are used in the inventive composition, these can be used with the carrier matrix in subconfluent state. At this time they are in their optimal growth phase, yet share very common and respond very well to the cytokines that are produced by the gene-transfected fibroblasts.

The inventive composition may also keratinocytes, in a preferred Ausführungsfor specifically include preferably autologous keratinocytes. The keratinocytes are in particular the outer layer of skin, called epidermis. Keratinocyte cultures can be cultivated by a known routine technique [eg Rheinwald et al. Nature 265 (1977) pp 421-424]. In this case, a small piece of skin is usually removed by biopsy and then epidermis and is separated from each other. From the epidermis, a cell suspension is prepared, preferably by treatment with a proteolytic enzyme. The thus-obtained individual cells are then grown in culture containers (flasks or dishes) under sterile conditions and replaced at an appropriate time of the cultivation vessels. The composition of the invention comprises genetically modified fibroblasts. Fibroblasts are the Mesenc ym entstammende cells with a large cell body and a slightly flattened core. The fibroblasts are particularly involved in the formation of the intercellular substance of connective tissue. According to the invention, either autologous fibroblasts can be used alone or in a preferred form of allogeneic fibroblasts are used by another individual, not come to the patient to be treated. Very particularly preferably those fibroblasts are used which were clonally selected, ie derived from a clone.

In a particularly preferred embodiment, the gene-transfected fibroblasts were treated with a dose of ionizing radiation such that the fibroblasts can no longer multiply, and die after a certain time. This radiation has the advantage that the gene-transfected cells die in the body, after a reasonable time (<3 weeks). Another advantage of the use of irradiated fibroblasts is that the so-treated cells continue to express the cytokine good.

In a particularly preferred embodiment, cells are immortalized fibroblast cell lines used by the present invention, wherein a cell line which has received the designation KMST-6, has proven particularly useful. Immortalized fibroblast cell lines are advantageous because they can be used continuously cultured and biologically well characterized. Without difficulty but other appropriate immortalized fibroblast cell lines can be used.

In the inventively used fibroblasts is a foreign gene that encodes a suitable cytokine, is introduced. It is preferable that in this foreign gene is a gene encoding a cytokine, such as EGF, TGF-α or KGF. The epidermal growth factor (epidermal growth factor) is abbreviated as EGF. This is a globular protein of about 6.2 kDa, which has 53 amino acids. According to the invention it is not essential that the complete gene is introduced into the transfected fibroblasts; it is sufficient if the part is introduced, which has the biological activity. To allow secretion of the biologically active peptide, the corresponding cDNA moiety is preferably fused in-frame with the secretory signal sequence of the human G-CSF. According to the invention are also preferred EGF-like proteins, such as transforming growth factor

(Transforming growth factor), TGF-α, which has a high homology to EGF, are used. The biological activity of EGF and TGF-α is comparable. Both cytokines have an effect on the epidermal development and differentiation of the cells.

may preferably be used also NGF (nerve growth factor). This cytokine is mainly responsible for the survival, differentiation and functional activity of sensory and sympathetic neurons in the peripheral nervous system. The accelerating effect on wound healing may be due to the ability of NGF to increase the survival and functional activities of various immunocompetent cells, such as granulocytes, mast cells, macrophages and Ly phozyten.

Another preferred cytokine is employed the keratinocyte growth factor (KGF), which stimulates in particular the division of proliferating keratinocytes. According to the invention, variants of the genes may be employed. Such variants may comprise deletions or attachments, and the sequence can be specifically altered. It is also quite possible to use such variants of cytokines, which have a higher biological activity as the naturally occurring cytokines, such as in particular fusion constructs of two or more cytokines. The fibroblasts used in this invention contain a foreign gene coding for a wound-healing promoting cytokine. This foreign gene may preferably be human, but also from an animal such as a mouse or bovine derived. However, a prerequisite is that the cytokine is then not species specific, when the foreign gene from another species originated.

It is necessary that the foreign gene is introduced into fibroblasts. This can be effected in that the desired gene is incorporated into a suitable vector and then transfected into the fibroblasts. Suitable vectors are viral vectors or plasmid vectors, the plasmid vectors are especially preferred as must be carried out in viral vectors additional tests to rule out contamination with replication-competent viruses.

The compositions of the invention have several advantages over the methods known from the prior art solutions.

By genetically modified fibroblasts, which were irradiated in a preferred embodiment lethal, can be expressed or the desired cytokines for a predetermined time and released into the tissue. Due to the continuous production of the cytokine, the problems can be avoided, due to the short half-life of cytokines in the local external application. In the inventive compositions, different types of cells (keratinocytes / fibroblasts) can be combined with each other, wherein the gene-transfected fibroblasts can also include different cytokines. Thereby, various growth factors can be introduced into the wound area targeted. If autologous keratinocytes are used, the Histoinkompatibilitätsabstoßungsreaktion can be avoided. It is also possible to additionally introduce genes for growth factors that act on white blood cells, such as G-CSF or GM-CSF, to thereby stimulate the body's immune system into the wound areas. Thus, the body's defenses can be strengthened to infection, which is especially relevant for burn wounds.

It is also quite possible to use the composition according to the invention without keratinocytes. In this case, the cytokine is released into the environment by genetically modified fibroblasts and keratinocytes are stimulated those of the treated patients are found for example in the peripheral areas of the wound.

The compositions of the invention are preferably in the form of pharmaceutically acceptable formulations. This can be either suspensions of fibrin glue and gene-transfected fibroblasts, which can be applied as solutions, suspensions, ointments, or in the form of gels on the wound. If it is a composition which are a fixed component, ie having a carrier matrix then these compositions preferably in the form of sterile units, which are conserved in a suitable manner. Such preparations may be, for example, in the form of deep-frozen sterile conditions.

Description of the Figure

Figure 1 shows the structure of the expression vector for human EGF. Part (A) shows the plasmid with the chimeric EGF gene. Part (B) shows the sequence of in-frame fusion between the human G-CSF signal sequence (underlined) and the mature coding for human EGF region (Asn Ser Asp ...).

Figure 2 shows the secretion of EGF by fibroblasts transfected with the EGF-Plas id pCMV-IRES-TKNeo. This is to fibroblast cell line KMST-6 after irradiation (100 Gy). In the experiment, 2 x 10 were seeded irradiated cells in culture plates with six holes and the EGF concentration was determined in culture supernatants after 24 hours. Values ​​represent averages.

Figure 3 shows the bioactivity of EGF chimeric polypeptides obtained from clones # 3 and # 6 of gene-transfected KMST-6 fibroblasts.

Part (A) shows the bioactivity of Mäusekeratinozyten from BALB / MK.

Part (B) shows the results obtained with the aid of human primary keratinocytes. The figures indicate the average results of eight measurements with standard deviation. The dotted bars represent the standard curve obtained with recombinant EGF. The open bars represent control values ​​obtained with fibroblasts of the cell line-KMST. 6 The striped and gray bars represent the results obtained with culture supernatants of both clones 3 and 6 respectively.

Figure 4 shows the proliferation of primary human keratinocytes in culture test with irradiated fibroblasts of the cell line KMST-6, which were gentransfiziert.

Part (A) shows the results after four days of co-culture.

Part (B) shows the results after 10 days of co-culture.

The present invention is further illustrated by the following examples:

example 1

Construction of a plasmid containing a gene for EGF and transfection in most human cells is synthesized as a 130 kDa precursor molecule Transme branglycoprotein-EGF, which is proteolytically cleaved in the biologically active EGF 6.2 kDa peptide of 53 amino acids. According to the invention, therefore, a chimeric construct was prepared which codes for an in-Leserah en fusion of the mature EGF peptide and human G-CSF secretory signal sequence. The structure is shown schematically in Figure 1 (A). The thereby resulting DNA fusion fragment was placed under the transcriptional control of the human CMV (cytomegalovirus) promoter and incorporated into a dicistronic vector to bind the expression of the transgene to the selection marker neomycin transferase phosphorus.

In cloning the DNA encoding the mature human EGF peptide sequence was amplified by PCR technology, and the product thereby obtained in the vector pBluescript (Stratagene) and then sequenced. Similarly, the human G-CSF signal sequence was amplified from human G-CSF cDNA by PCR technology, in which an improved Kozak consensus sequence at the 5 'end and a single Nhel restriction site was created. The relevant sequence is shown in Figure 1 (B).

The plasmid thus generated was transfected into human fibroblast cell line KMST-6 and all neomycin-resistant clones secreted human EGF, which was detected by an ELISA assay. The control showed that untransfected human fibroblasts of the cell line KMST-6 secreted no detectable amounts of hEGF, both before and after irradiation.

The clones were # 3 and # 6 are used for the further investigations, the secreted 37 and 8 ng EGF / 10 6 cells and 24 hours. The secretion of human EGF in the supernatant by transfected fibroblasts was determined using the ELISA technique (Quantikine, R & D Systems). the supernatant from irradiated or non-irradiated output cells or EGF gene-transfected cells, it was taken out after 24 hours and tested for the EGF production, after the number of viable cells was determined.

Irradiation was performed at room temperature with a 137Cs- radiation source at a dose rate of 3 Gy / minute.

example 2

Influence of lethal irradiation on the expression of EGF chimeric protein by fibroblasts gentransf ied

For optimal wound healing results, it is necessary that EGF is on sale in the first days of treatment. On the other hand, can be prevented by lethal irradiation, the uncontrolled in vivo growth of genetically modified cells. the influence of lethal irradiation on the expression of EGF It was therefore investigated, wherein KMST-6 fibroblasts (clone # 3, transfected with the plasmid pCMV-EGF-IRES-TKNeo) were examined. It was found that after irradiation with 100 Gy the secretion of EGF slowly dropped, but at least seven days was detectable in the supernatant in vitro. The results are summarized in Fig. 2

example 3

Biological activity of chimeric EGF polypeptide

In order to demonstrate that the EGF protein, which is secreted by the transfected fibroblasts, actually having the biological activity, the supernatants of the media were checked by the EGF gene-transfected clones on the mitogenic activity of permanent Mäusekeratinozyten cell lines and primary human keratinocytes , To control various concentrations of recombinant human EGF were employed. Both cell types were stimulated in a dose dependent manner by both the recombinant protein as well as by the culture supernatants of clones # 3 and # 6. An optimal stimulation of Mäusekeratinozyten was used at a concentration between 2 and 20 ng / ml of recombinant hEGF observed. This corresponds to the stimulation, accessible by a 1: 5 dilution of culture supernatant. The results are shown in Figure 3 (A).

When using primary human keratinocytes, the effective dose was slightly lower and there was an optimal proliferation already at 0.2 ng / ml rEGF and at a 1:50 dilution of the culture supernatant of the clone pCMV-EGF-IRES TKNeo / KMST6 # 3 observed. The results are shown in Figure 3 (B). In both tests, the tendency was observed that the Zellwachstu was inhibited at higher concentrations. Culture supernatants from non-transfected fibroblasts of the cell line KMST-6 showed no stimulation of the proliferation in comparison with cell culture supernatants without added growth factor. In order to investigate the therapeutic efficacy in wound healing in more detail, were lethally irradiated EGF gene transfected fibroblasts in vitro co-cultured with primary human keratinocytes. In the experiments, it was found that a dose dependent stimulation of keratinocyte proliferation that was similar after an incubation period of four days with various concentrations of EGF irradiated secreting fibroblasts are obtained, which was induced by the recombinant growth factor. This is illustrated in Figure 4, wherein Figure 4 (A) shows the values ​​after four days of co-cultivation and the Figure 4 (B) shows the values ​​after 10 days of co-cultivation. example 4

Detection of in vivo EGF production by in vitro liposomal transfected KMST-6 cells

Versuchsauf construction

1.5 x 1.5 cm full-thickness skin wounds were created on the back of 42 nude mice. 9.4 x 10 hEGF-transfected and lethally irradiated KMST-6 cells were suspended in 2.8 ml of fibrin glue and on the full-thickness skin wounds of 14 nude mice

2 transplanted (300 000 cells / cm, Group I).

14 were full-thickness skin wounds than controls transplanted with untransfected KMST-6 cells (300,000 cells / cm 2, Group II) and 14 untreated whole skin wounds (Group III).

On day 1, 2, 3, 4, 5, 7 and 14 respectively, two animals were necropsied from each group and homogenized 0.8 g wound tissue with a Triton X-PBS buffer. The homogenates were centrifuged and analyzed the EGF concentration in the supernatants by means of an anti-human EGF ELISA.

.Results

In vivo were in group I (Invention) day 1 470 pg / ml detected, compared with 18 pg / ml in group II and 1.3 pg / ml in group III. On days 2-7, the EGF concentrations decreased in group I, but were significant higher than in the control groups. On day 14, no EGF was detectable in all three groups.

In summary, these results indicate that in vitro EGF-transfected fibroblasts were successfully transplanted into a fibrin glue suspension. The transgenic protein was detectable at least until day 7 in vivo. The results obtained in the experiment are summarized in Table I below.

table I.

Days pCMV-EGF-IRES KMST6 untreated TKNeo / KMST6 # 3 control Vollhaut¬

(Transplanted wounds according to the invention with untransfected (group) III

KMST6 cells

Group II)

1 470 18 1.3

2 393 58 1.6

3330 28 2.3

4150 8 6.5

5180 8.5 8

7140 8.3 2.6

14 0.4 0 0

Table I shows the release hEGF in pg / ml in vivo in wounds by irradiated KMST6 fibroblasts which have been transfected with chimeric hEGF gene.

example 5

Wound healing promoting effect after transplantation of in vitro EGF -transf izierten KMST-6 cells in combination with human keratinocytes (mixed cell transplantation)

experimental setup

1.5 x 1.5 cm full-thickness skin wounds were created on the back of 72 nude mice. 1.0125 x 10 EGF-transfected and lethally irradiated KMST-6 cells were suspended in combination with 2.025 x 10 human keratinocytes in 3.6 ml of fibrin glue and on full-thickness skin wounds of 18 nude mice transplanted (Ratio 1: 2, 75 000 cells / cm 2 , group I). As a control, were 18 full thickness wounds, transplanted with EGF-transfected KMST-6 cells alone (25,000 cells / cm, Group II), 18 full-thickness skin wounds with transplanted humanener

2

Keratinocytes alone (50,000 cells / cm, group III) as well as 18 full-thickness skin wounds transplanted with non-transfected KMST-6

Cells in combination with human keratinocytes (Ratio 1: 2, 7755 000000 ZZeelllleenn cc // 2, Group IV). Table II shows the group division.

table II

On day 1, 3, 5, 7, 10 and 12, one animal from each group was necropsied and 0, 8g wound tissue was homogenized with a Triton X-PBS buffer. The homogenates were centrifuged and analyzed the EGF concentration of the supernatants using an anti-human EGF ELISA. The other half of the biopsies was starting with day 5 used for histological examinations.

each biopsies of two additional animals from each group were examined histologically at the post-surgical days 7, 10, 12, 14, 17 and 21st

Results

It was detectable in the group I and II EGF but not in group III and IV.

Regarding the wound closure, the results continually showed almost complete, distinct, from 14 days a complete epithelialization in Group I at day 7 to 12th The best results in terms of Rekonstitutionsqualitat the epidermis (Zellagigkeit the epithelium) also showed the wounds of Group I. The control groups showed against it until the day 14 a pronounced epithelialization and on day 21 they still showed no quality epithelialization. In Table III, the epithelialization is tabulated.

Table III: Tabular presentation of epithelialization

Epithelialization / differentiation

no detectable epithelialization + incipient epithelialization

++ strong but not complete epithelialization +++ complete epithelialization

example 6

Wound healing promoting effect after transplantation of izierten in vitro EGF-transf KMST-6 cells in a large animal model

experimental setup

In a large animal experimental tests on a total of 3 pigs (Pl, P2, P3) with a total of 21 each were standardized

Burn wounds (5 cm 2 in size, degree 2a) is performed. In one pig (Pl) 7 standardized burn wounds with EGF-transfected KMST-6 cells were transplanted in fibrin glue (therapy group). Here served 7 untreated standardized burn wounds (control group I) and 7 standardized burn wounds treated with fibrin glue (control group III), as a control. In two other pigs (P2, P3) also each 7 standardized burn wounds with EGF-transfected KMST-6 cells were transplanted in fibrin glue (therapy group). 7 each untreated standardized burn wounds (control group I), and each 7 standardized burn wounds that have been transplanted with non-transfected cells KMST-6 (control group II) served as controls. Table IV shows the group division.

table IV

On day 1, 3, 5, 7, 10, 21 and 35 are each a wound was biopsied from each group. 0.8 g of the wound tissue biopsies to day 10 were homogenized with a Triton X-PBS buffer. The homogenates were centrifuged and analyzed the EGF concentration of the supernatants using an anti-human EGF ELISA. Furthermore, the biopsies were evaluated histologically.

Results

Table V shows EGF concentration values ​​in each of a wound of the groups. Table V EGF-tissue concentrations in pg / ml in the coarse animal experiment

Claims

claims
1) A composition for treatment of wounds, the fibroblasts containing at least one foreign gene containing coding for a wound-healing promoting cytokine and at least one further comprising promoting wound healing component.
2) Composition according to claim 1, characterized in that the wound healing-promoting component is a fibrin glue.
3) A composition according to claim 2, characterized in that the fibrin fibrinogen and fibronectin, as well as given alls albumin, factor XIII, and Plasminogen comprises.
4) Composition according to claim 1, characterized in that the further promoting wound healing component is a solid component which serves as a support matrix.
5) Composition according to claim 4, characterized in that the solid component is a made of a Hyaluronsäureester support matrix.
6) Composition according to any one of the preceding claims, characterized in that it also comprises keratinocytes.
7) A composition according to any one of the preceding claims, characterized in that the gene encoding the cytokine is selected from the group comprising the gene factor encoding transforming growth α (TGF-α), the gene factor encoding the epidermal growth factor (EGF ), the gene coding for the basic fibroblast growth factor (b-FGF), the gene encoding nerve growth factor (NGF) and the gene encoding the keratinocyte growth factor (KGF). 8) A composition according to any one of the preceding claims, characterized in that the fibroblasts comprises at least one further foreign gene coding for a cytokine that acts on blood cells.
9) A composition according to any one of the preceding claims, characterized in that the gene coding for the cytokine was introduced with the aid of a plasmid vector in the fibroblasts.
10) A composition according to claim 9, characterized in that the plasmid vector having an in-reading frame of the fusion gene coding for the mature cytokine and a secretory signal sequence.
11) A composition according to claim 10, characterized in that it is in the secretory signal sequence to that of the human G-CSF gene.
12) A composition according to any one of the preceding claims, characterized in that it is autologous fibroblasts.
13) A composition according to any one of claims 1 to 11, characterized in that it is allogeneic fibroblasts.
14) A composition according to any one of the preceding claims, characterized in that the fibroblasts were irradiated.
15) A composition according to any one of the preceding claims, characterized in that it is clonally-selected fibroblasts.
16) A composition according to any one of claims 13 to 15, characterized in that it is in the allogeneic fibroblasts fibroblasts of the cell line-KMST. 6 17) Use of fibroblasts that at least one foreign gene encoding a cytokine contain wound healing, for the manufacture of a medicament for the treatment of wounds.
18) Use according to claim 17, characterized in that the foreign gene is selected among the genes encoding the epidermal growth factor (EGF), transforming
Growth factor α (TGF-α), nerve growth factor, basic fibroblast growth factor (b-FGF) and keratinocyte growth factor (KGF).
PCT/EP1998/002038 1997-04-17 1998-04-08 Fibroblasts with a foreign gene-containing composition for treating wounds WO1998048012A1 (en)

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JP54493298A JP2002501491A (en) 1997-04-17 1998-04-08 Wound healing compositions comprising fibroblast cells with a foreign-heterologous gene
CA 2286548 CA2286548A1 (en) 1997-04-17 1998-04-08 Fibroblasts with a foreign gene-containing composition for treating wounds
EP19980919236 EP0975756A1 (en) 1997-04-17 1998-04-08 Fibroblasts with a foreign gene-containing composition for treating wounds

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US7846465B1 (en) 1999-05-28 2010-12-07 Fibrocell Science, Inc. Method of using autologous fibroblasts to promote healing of wounds and fistulas
US20140178346A1 (en) * 2012-12-26 2014-06-26 The Regents Of The University Of California Cellular compositions for tissue engineering

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