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WO1998048012A1 - Fibroblastes avec une composition contenant un gene etranger pour traiter des blessures - Google Patents

Fibroblastes avec une composition contenant un gene etranger pour traiter des blessures Download PDF

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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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WO
WIPO (PCT)
Prior art keywords
fibroblasts
composition according
growth factor
gene coding
egf
Prior art date
Application number
PCT/EP1998/002038
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German (de)
English (en)
Inventor
Roland Mertelsmann
Felicia Rosenthal
Peter Kulmburg
Björn G. STARK
Eszter Tanczos
Jürgen Kopp
Original Assignee
KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG filed Critical KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG
Priority to EP98919236A priority Critical patent/EP0975756A1/fr
Priority to JP54493298A priority patent/JP2002501491A/ja
Priority to CA002286548A priority patent/CA2286548A1/fr
Priority to IL13163698A priority patent/IL131636A0/xx
Priority to AU72150/98A priority patent/AU739125B2/en
Publication of WO1998048012A1 publication Critical patent/WO1998048012A1/fr
Priority to NO995054A priority patent/NO995054D0/no

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to a composition for the treatment of damage to the skin which is difficult to treat.
  • it can be a considerable problem to restore the skin areas destroyed by the damage, in particular if predominant parts of the skin are severely damaged.
  • other diseases such as tumor diseases or chronic skin damage, it is often a very significant problem to restore the damaged skin areas.
  • 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 purpose of this application is to treat genetic defects in the human genome.
  • a functionally active "replacement" gene is introduced into a somatic cell and used to compensate for the damage caused by the defective gene. This reference does not address the treatment of wounds or the
  • Cytokines that promote wound healing such as TGF- ⁇ , EGF or b-FGF.
  • the "fibroblast growth factor” mentioned in claim 5 is used as a substance forming blood vessels.
  • keratinocyte growth factor acting on keratinocytes
  • the subject of WO 90/08771 is the genetic engineering provision of growth factor proteins which influence epitelial cells.
  • the human keratinocyte growth factor is used as an essentially pure protein.
  • U.S. Patent 5,302,701 describes the development of an artificial polypeptide that is both cell adhesive and has cell growth promoting activity. It is a combination of fibronectin and Fibroblast Growth Factor (FGF).
  • FGF Fibroblast Growth Factor
  • US Pat. No. 5,196,196 relates to a wound dressing which also comprises a carrier matrix, there is an essential difference from the present invention with regard to the principle of action and the active agent.
  • purified protease Nexin-I (PN-I) is used for the wound dressing. However, this is not a growth factor, but an enzyme with certain properties.
  • the protease Nexin-I is a serine protease inhibitor; a member of the serine protease superfamily that is synthesized and secreted by human fibroblasts in culture.
  • the protein is used in a purified form and not in the form of a gene which is expressed by fibroblasts into which this gene has been incorporated.
  • keratinocyte preparations for example so-called keratinocyte sheets
  • keratinocyte sheets which are very difficult to handle and can often only be used when spatial structures have formed during cell cultivation.
  • allogeneic preparations in particular those which come from different donors and are not characterized are contaminated with viruses (HIV, HCV or viruses that have not yet been characterized) and that the patient to be treated can thereby be infected.
  • the present invention therefore relates to compositions for the treatment of wounds containing fibroblasts which contain at least one foreign gene coding for a cytokine which promotes wound healing and at least one further component which promotes wound healing.
  • the composition according to the invention can have further constituents which are usually used in such compositions.
  • the further component that promotes wound healing can be a so-called fibrin glue.
  • Fibrin adhesives of this type are commercially available and are used in various fields of medicine, in particular in surgery.
  • the fibrin glue contains fibrinogen, which is converted from thrombin to monomeric fibrin. This in turn forms aggregated fibrin through end-to-end or side-to-side attachment.
  • the fibrin glue also contains fibronectin.
  • thrombin activates factor XIII, which is usually present in sufficient quantities in fibrin glue.
  • the fibrin glue contains a thrombin solution as a further component, which is often added together with calcium chloride as a separate component.
  • the fibrin glue can also contain sufficient amounts of albumin or plasminogen.
  • the further component that promotes wound healing can also be a solid component that serves as a carrier matrix.
  • Carriers for "artificial skin” are commercially available (for example Laserskin® or Biobrane®).
  • the solid components can preferably be a matrix of a derivative of hyaluronic acid, preferably a hyaluronic acid ester.
  • Hyaluronic acid is an endogenous component in the connective tissue that is subject to an extremely high turnover rate. If the matrix consists of hyaluronic acid, it is quickly broken down in the wound by the body's own hyaluronidase. Therefore, according to the invention, preference is given to using derivatives of hyaluronic acid which are broken down somewhat more slowly in the body.
  • a particular advantage that can be achieved by using a carrier matrix is that cells can be applied to the wound that do not yet form a cell cluster that has grown together. If keratinocytes are also used in the composition according to the invention, these can be used in the subconfluent state with the aid of the carrier matrix. At this point, they are in their optimal growth phase, still divide very often and react particularly well to the cytokines that are produced by the gene-transfected fibroblasts.
  • the composition according to the invention can also comprise keratinocytes, specifically preferably autologous keratinocytes.
  • the keratinocytes form in particular the outer skin layer, the so-called epidermis.
  • Keratinocyte cultures can be cultivated according to a routine technique known per se [eg Rheinwald et al. Nature 265 (1977) pp. 421-424]. Usually a small piece of the skin is removed by biopsy and then the epidermis and the der is separated. A cell suspension is produced from the epidermis, preferably by treatment with a proteolytic enzyme. The individual cells thus obtained are then grown in culture containers (bottles or dishes) under sterile conditions and detached from the cultivation containers at a suitable time.
  • the composition according to the invention has genetically modified fibroblasts.
  • Fibroblasts are cells from the mesenc ym with a large cell body and a somewhat flattened nucleus.
  • the fibroblasts are particularly involved in the formation of the intercellular substance of the connective tissue.
  • autologous fibroblasts can be used or, in a preferred form, allogeneic fibroblasts are used which originate from another individual, ie not from the patient to be treated.
  • Those fibroblasts that have been selected clonally, that is, derived from a clone, are very particularly preferably used.
  • the gene-transfected fibroblasts have been 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 in the body die after a reasonable time ( ⁇ 3 weeks).
  • Another advantage of using irradiated fibroblasts is that the cells treated in this way continue to express the cytokine well.
  • cells of immortalized fibroblast cell lines are used according to the invention, a cell line which has been given the name KMST-6 having proven particularly useful.
  • Immortalized fibroblast cell lines are advantageous because they can be continuously cultivated and used in a biologically precise manner.
  • other suitable immortalized fibroblast cell lines can also be used without difficulty.
  • a foreign gene which codes for a suitable cytokine is introduced into the fibroblasts used according to the invention.
  • This foreign gene is preferably a gene which codes for a cytokine such as EGF, TGF- ⁇ or KGF.
  • the epidermal growth factor is briefly referred to as EGF. It is a globular protein of approximately 6.2 kDa, which has 53 amino acids. According to the invention, it is not absolutely necessary for the complete gene to be introduced into the transfected fibroblasts; it is sufficient if the part which has the biological activity is introduced.
  • the corresponding cDNA portion is preferably fused in-reading frame with the secretory signal sequence of the human G-CSF.
  • EGF-like proteins such as the transforming growth factor, are also preferred according to the invention
  • TGF- ⁇ (transforming growth factor) TGF- ⁇ , which has a high homology to EGF.
  • the biological activity of EGF and TGF- ⁇ is comparable.
  • Both cytokines have an influence on the epidermal development and the differentiation of the cells.
  • NGF nerve growth factor
  • This cytokine is primarily 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 lymphocytes.
  • cytokine is the keratinocyte growth factor (KGF), which in particular stimulates the division of keratinocytes that are capable of division.
  • KGF keratinocyte growth factor
  • Variants of the genes can also be used according to the invention. Such variants can have deletions or additions and the sequence can be changed in a targeted manner. It is also entirely possible to use variants of cytokines which have a higher biological activity than the naturally occurring cytokines, such as, in particular, fusion constructs of two or more cytokines.
  • the fibroblasts used according to the invention contain a foreign gene which codes for a cytokine which promotes wound healing. This foreign gene can preferably come from humans, but also from an animal, such as, for example, mice or cattle. However, the prerequisite is that the cytokine is not species-specific if the foreign gene comes from another species.
  • the foreign gene is required to be introduced into the fibroblasts. This can be accomplished by incorporating the desired gene into a suitable vector and then transfecting it into the fibroblasts.
  • Viral vectors or plasmid vectors are suitable as vectors, the plasmid vectors being particularly preferred, since additional tests have to be carried out on viral vectors to rule out contamination with replication-competent viruses.
  • compositions according to the invention have various advantages over the solutions known from the prior art.
  • the gene-modified fibroblasts which in a preferred embodiment were lethally irradiated, can express the desired cytokine or s for a predetermined time and release them into the tissue. Due to the continuous production of the cytokine, the problems that can be attributed to the short half-life of cytokines with local external application can be avoided.
  • different cell types keratinocytes / fibroblasts
  • the gene-transfected fibroblasts can also contain different cytokines. This enables various growth factors to be introduced into the wound area in a targeted manner. If autologous keratinocytes are used, the histoin incompatibility rejection reaction can be avoided.
  • composition according to the invention without keratinocytes.
  • the gene-modified fibroblasts release the cytokine into the environment and stimulate those keratinocytes of the treated patient that are found, for example, in the marginal areas of the wound.
  • compositions according to the invention are preferably in the form of pharmaceutically acceptable formulations. These can either be suspensions of fibrin glue and gene-transfected fibroblasts, which can be applied to the wound as solutions, suspensions, ointments or in the form of gels. If it is a composition which has a solid component, that is to say a carrier matrix, then these compositions are preferably in the form of sterile units which are preserved in a suitable manner. Such preparations can, for example, be in the form of frozen, sterile pads.
  • Figure 1 shows the structure of the expression vector for human EGF.
  • Part (A) shows the plasmid construction with the chimeric EGF gene.
  • Part (B) shows the sequence of the fusion in the reading frame between the human G-CSF signal sequence (underlined) and the mature region coding for human EGF (Asn Ser Asp ).
  • FIG. 2 shows the secretion of EGF from fibroblasts which were transfected with the plas id pCMV-EGF-IRES-TKNeo. These are fibroblasts from the KMST-6 cell line after irradiation (100 Gy). In the experiment, 2 x 10 were irradiated Cells were seeded in six well culture plates and the EGF concentration was determined in culture supernatants after 24 hours. The values represent average values.
  • Figure 3 shows the bioactivity of EGF chimeric polypeptides obtained from clones # 3 and # 6 from gene transfected KMST-6 fibroblasts.
  • Part (A) shows the bioactivity on mouse keratinocytes from the BALB / MK strain.
  • Part (B) shows the results obtained using human primary keratinocytes.
  • the values give the average results of eight measurements with standard deviation.
  • the dotted bars represent the calibration curve obtained with recombinant EGF.
  • the empty bars represent the control values obtained with fibroblasts from the KMST-6 cell line.
  • the striped and gray bars represent the results obtained with culture supernatants from both clones 3 and 6, respectively.
  • FIG. 4 shows the proliferation of primary human keratinocytes in the culture test with irradiated fibroblasts of the KMST-6 cell line, which were gene-transfected.
  • Part (A) shows the results after four days of co-culture.
  • Part (B) shows the results after 10 days of co-culture.
  • EGF is synthesized as a 130 kDa transme branglycoprotein precursor molecule that is proteolytically cleaved into the biologically active 6.2 kDa EGF peptide with 53 amino acids.
  • a chimeric construct was therefore produced which codes for an in-reading fusion of the mature EGF peptide and the human G-CSF secretory signal sequence.
  • the structure is shown schematically in Figure 1 (A).
  • the resulting DNA fusion fragment was placed under the transcriptional control of the human CMV (cytomegalovirus) promoter and inserted into a dicistronic vector in order to bind the expression of the transgene to that of the selection marker neomycin phosphorus transferase.
  • CMV cytomegalovirus
  • the sequence coding for the mature human EGF peptide was amplified with the aid of PCR technology and the product obtained was subcloned in the vector pBluescript (Stratagene) and then sequenced.
  • the human G-CSF signal sequence from human G-CSF cDNA was amplified using PCR technology, creating an improved Kozak consensus sequence at the 5 'end and a single Nhel restriction site. The relevant sequence is shown in Figure 1 (B).
  • the plasmid thus generated was transfected into human fibroblasts of the KMST-6 cell line and all neomycin-resistant clones secreted human EGF, which was demonstrated by an ELISA test.
  • the control showed that non-transfected human fibroblasts from the KMST-6 cell line did not secrete detectable levels of hEGF either before or after radiation.
  • Irradiation was carried out at room temperature with a 137 CS radiation source at a dose rate of 3 Gy / minute.
  • EGF For optimal wound healing results, EGF must be available for the first few days of treatment.
  • lethal radiation can prevent the uncontrolled in vivo growth of genetically modified cells.
  • the influence of lethal radiation on the expression of EGF was therefore investigated, KMST-6 fibroblasts (clone # 3, transfected with the plasmid pCMV-EGF-IRES-TKNeo) being examined. It was found that EGF secretion slowly decreased after irradiation with 100 Gy, but was detectable in the supernatant for at least seven days in vitro. The results are summarized in Figure 2.
  • Table I shows the hEGF release in pg / ml in vivo in wounds from irradiated KMST6 fibroblasts transfected with the chimeric hEGF gene.
  • Keratinocytes alone (50,000 cells / cm, group III) and 18 whole skin wounds transplanted with non-transfected KMST-6
  • the results in group I continuously showed an almost complete, pronounced epithelialization on day 7 to 12, and a complete epithelialization on day 14.
  • the wounds also showed the best results with regard to the reconstitution quality of the epidermis (cell activity of the epithelium) of group I.
  • the control groups on the other hand, only showed pronounced epithelialization on day 14 and on day 21 they still showed no high-quality epithelialization.
  • Table III shows the epithelialization in a table.
  • Burn wounds (5 cm 2, grade 2a) performed.
  • a pig 7 standardized burns with EGF-transfected KMST-6 cells were transplanted into fibrin glue (therapy group).
  • 7 untreated standardized burns control group I
  • 7 standardized ones were used here Burns treated with fibrin glue (control group III) as a control.
  • two other pigs P2, P3
  • 7 standardized burns with EGF-transfected KMST-6 cells were transplanted into fibrin glue (therapy group).
  • 7 untreated standardized burn wounds control group I
  • 7 standardized burn wounds which were transplanted with non-transfected KMST-6 cells (control group II), served as controls.
  • Table IV shows the group breakdown.
  • one wound was biopsied from each group.
  • 0.8g of wound tissue from the biopsies up to day 10 were homogenized with a Triton-X-PBS buffer.
  • the homogenates were centrifuged and the EGF concentration of the supernatants was evaluated using an anti-human EGF ELISA.
  • the biopsies were also evaluated histologically.
  • Table V shows EGF concentration values in one wound each of the groups.
  • Table V EGF tissue concentrations in pg / ml in the large animal experiment

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Abstract

Cette composition pour traiter des blessures comprend des fibroblastes qui contiennent au moins un gène étranger de codage d'une cytokine qui favorise la guérison de blessures et au moins un autre élément constitutif qui favorise la guérison de blessures.
PCT/EP1998/002038 1997-04-17 1998-04-08 Fibroblastes avec une composition contenant un gene etranger pour traiter des blessures WO1998048012A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP98919236A EP0975756A1 (fr) 1997-04-17 1998-04-08 Fibroblastes avec une composition contenant un gene etranger pour traiter des blessures
JP54493298A JP2002501491A (ja) 1997-04-17 1998-04-08 外来・異種遺伝子を有する繊維芽細胞を含んでなる創傷治癒組成物
CA002286548A CA2286548A1 (fr) 1997-04-17 1998-04-08 Fibroblastes avec une composition contenant un gene etranger pour traiter des blessures
IL13163698A IL131636A0 (en) 1997-04-17 1998-04-08 Fibroblasts with a foreigh gene-containing composition for treating wounds
AU72150/98A AU739125B2 (en) 1997-04-17 1998-04-08 Composition for treating wounds which comprises fibroblasts harboring a foreign gene
NO995054A NO995054D0 (no) 1998-04-08 1999-10-15 Sammensetning til sårbehandling, omfattende fibroblaster inneholdende fremmede gen

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19716098.0 1997-04-17
DE19716098A DE19716098A1 (de) 1997-04-17 1997-04-17 Fibroblasten mit einem Fremdgen enthaltende Zusammensetzung zur Behandlung von Wunden

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WO1998048012A1 true WO1998048012A1 (fr) 1998-10-29

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EP (1) EP0975756A1 (fr)
JP (1) JP2002501491A (fr)
AU (1) AU739125B2 (fr)
CA (1) CA2286548A1 (fr)
DE (1) DE19716098A1 (fr)
IL (1) IL131636A0 (fr)
WO (1) WO1998048012A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6277368B1 (en) 1996-07-25 2001-08-21 The Regents Of The University Of California Cancer immunotherapy using autologous tumor cells combined with cells expressing a membrane cytokine
JP2004531453A (ja) * 2000-05-24 2004-10-14 ウニベルジテートスクリニクム フライブルク トランスフェクション系
CN115245561A (zh) * 2021-04-25 2022-10-28 浙大城市学院 三元复合物、制备方法、用途及用于角膜损伤的生长因子及其编码基因共载复合物滴眼液

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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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WO1985004185A1 (fr) * 1984-03-20 1985-09-26 Caplan Arnold I Procede et materiau pour stimuler la croissance de tissus cartilagineux et osseux en des sites anatomiques
EP0213908A2 (fr) * 1985-08-26 1987-03-11 Hana Biologics, Inc. Tissu artificiel transplantable et procédé
DE4406073A1 (de) * 1994-02-24 1995-08-31 Univ Ludwigs Albert Verfahren zur Herstellung von humanen, klonogenen Fibroblasten, Verfahren zur Gentransfizierung von Fibroblasten und so erhaltene Fibroblasten

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US5196196A (en) * 1986-06-03 1993-03-23 Incyte Pharmaceuticals, Inc. Use of protease nexin-I in wound dressings
WO1990008771A1 (fr) * 1989-01-31 1990-08-09 Rubin Jeffrey S Adn codant un facteur de croissance specifique contre des cellules epitheliales
WO1992015676A1 (fr) * 1991-03-08 1992-09-17 The Salk Institute For Biological Studies Therapie genique par les cellules somatiques
JP2829405B2 (ja) * 1991-10-14 1998-11-25 寳酒造株式会社 機能性ポリペプチド
JPH09504786A (ja) * 1993-09-07 1997-05-13 シドニー キンメル キャンサー センター ハプロタイプ適合サイトカイン分泌細胞および免疫応答を刺激するための使用方法

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WO1985004185A1 (fr) * 1984-03-20 1985-09-26 Caplan Arnold I Procede et materiau pour stimuler la croissance de tissus cartilagineux et osseux en des sites anatomiques
EP0213908A2 (fr) * 1985-08-26 1987-03-11 Hana Biologics, Inc. Tissu artificiel transplantable et procédé
DE4406073A1 (de) * 1994-02-24 1995-08-31 Univ Ludwigs Albert Verfahren zur Herstellung von humanen, klonogenen Fibroblasten, Verfahren zur Gentransfizierung von Fibroblasten und so erhaltene Fibroblasten

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Title
ROSENTHAL ET AL: "PARACRINE STIMULATION OF KERATINOCYTES IN VITRO AND CONTINOUS DELIVERY OF EPIDERMAL GROWTH FACTOR TO WOUNDS IN VIVO BY GENETICALLY MODIFIED FIBROBLASTS TRANSFECTED WITH A NOVEL CHIMERIC CONSTRUCT", IN VIVO, vol. 11, no. 3, May 1997 (1997-05-01) - June 1997 (1997-06-01), pages 201 - 208, XP002075251 *
SVENSJÖ ET AL: "GROWTH FACTOR DELIVERY TO FULL-THICKNESS PORCINE WOUNDS WITH HEGF-TRANSFECTED FIBROBLASTS", SURGICAL FORUM, vol. 47, 1996, pages 715 - 717, XP002075250 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6277368B1 (en) 1996-07-25 2001-08-21 The Regents Of The University Of California Cancer immunotherapy using autologous tumor cells combined with cells expressing a membrane cytokine
US7264820B2 (en) 1996-07-25 2007-09-04 The Regents Of The University Of California Cancer immunotherapy using autologous tumor cells combined with cells expressing a membrane cytokline
US7364726B2 (en) 1996-07-25 2008-04-29 The Regents Of The University Of California Pharmaceutical composition for cancer treatment containing cells that express a membrane cytokine
JP2004531453A (ja) * 2000-05-24 2004-10-14 ウニベルジテートスクリニクム フライブルク トランスフェクション系
JP2008208145A (ja) * 2000-05-24 2008-09-11 Universitaetsklinikum Freiburg トランスフェクション系
CN115245561A (zh) * 2021-04-25 2022-10-28 浙大城市学院 三元复合物、制备方法、用途及用于角膜损伤的生长因子及其编码基因共载复合物滴眼液

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AU739125B2 (en) 2001-10-04
CA2286548A1 (fr) 1998-10-29
EP0975756A1 (fr) 2000-02-02
DE19716098A1 (de) 1998-10-22
AU7215098A (en) 1998-11-13
JP2002501491A (ja) 2002-01-15
IL131636A0 (en) 2001-01-28

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