WO2001019186A1

WO2001019186A1 - Composition and method for treating wounds

Info

Publication number: WO2001019186A1
Application number: PCT/US2000/025441
Authority: WO
Inventors: Gregory A. Helm; Son Won Byeon
Original assignee: University Of Virginia Patent Foundation
Priority date: 1999-09-14
Filing date: 2000-09-14
Publication date: 2001-03-22
Also published as: AU7585300A

Abstract

The present invention relates to a composition and method for inducing the repair of wounds. The method comprises contacting the wound with a controlled amount of a growth factor.

Description

Composition and Method for Treating Wounds

Field of the Invention

The present invention is directed to compositions and methods for enhancing the repair of chronic wounds and pressure ulcers. More particularly the present invention is directed to the controlled administration of basic Fibroblast Growth Factor (bFGF) and other growth factors to chronic wounds to enhance the repair of those wounds.

Background of the Invention

The treatment of chronic wounds and pressure ulcers is an enormous clinical problem, costing more than $6 billion/year in the US alone. Studies are currently underway evaluating the use of topically applied growth factors to improve healing of the lesions. However, proteins applied locally have short-term bioavailability and limited tissue penetration. These problems lead to limited tissue healing, and therefore, low efficacy. In an attempt to overcome the difficulties associated with the bioavailability of the growth factors, some have suggested treating chronic wounds by adding transgenic cells that constitutively express the growth factors (see US Patent No. 6,077,987). However, due to reports in the literature that the administration of excess amounts of growth factors could induce the formation of tumors, this approach is not favored. Since the application of growth factors is known to enhance wound healing, it is highly desirable that a method be developed for delivering controlled amounts of growth factors in a manner that allows adequate access to fibroblasts and other cells participating in the repair of the wound. The present invention is directed to overcoming the problems associated with the administration of growth factors to induce wound repair. In particular, the present invention is directed to compositions and methods for delivering a controlled supply of a modified version of bFGF to the site of a wound.

Summary of the Invention

The present invention is directed to a method of enhancing the healing of wounds by controlled delivery of exogenous growth factors to the wound site. In particular, the invention relates to the controlled expression of a gene, encoding a growth factor protein, at the site of a wound to provide a temporary and localized increased concentration of that growth factor, thus enhancing the repair of the wound.

Detailed Description of the Invention In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.

As used herein, "nucleic acid," "DNA," and similar terms also include nucleic acid analogs, i.e. analogs having other than a phosphodiester backbone. For example, the so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.

As used herein, "effective amount" means an amount sufficient to produce a selected effect. For example, an effective amount of bFGF is an amount sufficient to reduce the amount of time for a wound to heal relative to the absence of the growth factor.

As used herein, "operably linked" refers to a juxtaposition of elements to allow the elements to perform their usual function. For example, a promoter that is operably linked to a coding sequence means that the promoter is linked to the coding sequence in such a manner that the promoter is capable of effecting the expression of the coding sequence.

Pressure ulcers and other chronic wounds continue to be a major health care problem that affects large segments of the patient population, with particular impact on diabetics and immobilized individuals, such as the elderly or individuals with spinal cord injuries. It has been reported that the administration of purified growth factors to traumatized tissue stimulates vascularization and healing of burns, bone fractures, surgical abrasions such as those of plastic surgery, or wounds requiring repair. Furthermore, the use of topically applied growth factors to improve healing of lesions has been previously proposed. However, the benefits derived from such growth factor therapy have been limited. Growth factors, when applied locally have short-term bioavailability and limited tissue penetration, leading to limited tissue healing, and thus, low efficacy. In addition, there has been discussion that overstimulation of cellular growth through the use of growth factors could result in the formation of tumors. The present invention is directed to overcoming these problems/concerns by providing a carefully regulated supply of growth factors to the site of the wound.

The present invention is directed to a system for controlling the delivery of an effective amount of growth factor to a chronic wound site for repair of chronic wounds, including but not limited to pressure ulcers, poorly vascularized wound sites, and severe burns. The present method is based on the use of molecular regulatory signals to control the amount and timing of the delivery of the growth factor to the wound site. These regulatory sequences are operably linked to the coding sequence of growth factors to regulate the time, place and amount of expression of the growth factor. Regulatory elements suitable for use in accordance with the present invention are known to the skilled practitioner, and are selected from the group consisting of inducible promoters, upstream regulatory sequences, enhancers and the like. An inducible promoter for purposes of this invention includes any upstream regulatory element that increases expression of an operably linked gene when in the presence of the inducible agent.

One or more regulatory elements can be operably linked to the coding sequence of a growth factor to provide the desired regulated expression of the growth factor. In one preferred embodiment, the growth factor is linked to an inducible promoter such that expression of the growth factor takes place only in the presence of the inducing agent.

The growth factors suitable for use in the present invention are selected from the group consisting of Fibroblast Growth Factor (FGF), Platelet Derived Growth Factor (PDGF), Epidermal Growth Factor (EGF) and Tissue Growth Factor β (TGF-β). One or more of these growth factor can be administered as the active agent or the growth factor(s) can be combined with other wound healing compounds such as cytokines, including Interleukins such as Interleukin 4.

In one preferred embodiment basic Fibroblast Growth Factor is supplied to a wound site in a controlled manner to enhance the healing of the wound. Indications wherein bFGF is of value in encouraging neovascularization include musculo-skeletal conditions such as bone fractures, ligament and tendon repair, tendonitis, and bursitis; healing of wounds such as burns, cuts, lacerations, bed sores, and slow-healing ulcers such as those seen in diabetics; and in tissue repair during ischaemia and myocardial infarction.

To enhance the repair of wounds, the wound site is contacted with a wound healing composition comprising a nucleic acid sequence that encodes one or more preselected growth factors. The sequence encoding the growth factor is operably linked to regulatory sequences that allow for the controlled expression of the gene. Such regulatory sequences are know to those skilled in the art and include inducible promoters, upstream regulatory sequences, enhancers and the like. In this manner, the amount of growth factor can be tailored to provide an effective amount of growth factor to the cells surrounding the wound, based on the size, severity and type of wound. In addition, delivery of the growth factor can be limited to the most appropriate time during the healing process to enhance the healing process. The growth factor coding sequence is inserted into any of the eukaryotic expression vectors that are known to those skilled in the art to allow expression of the growth factor in a eukaryotic host cell. For example, one suitable expression vector is the pFlag-CMV vector (commercially available from Sigma Chemical Company 6050 Spruce Street, St. Louis, MO 63103).

One preferred growth factor for use in accordance with the present invention is basic Fibroblast Growth Factor (bFGF). The coding sequence of bFGF has been previously described (see US Patent No 5,604,293, the disclosure of which is incorporated herein). The entire bFGF coding sequence, or any biologically active fragment (i.e. capable of inducing neovascularization) thereof, can be used in accordance with the present invention. In one preferred embodiment, a nucleic acid sequence (SEQ ID NO: 1) encoding a truncated form of bFGF (thelδkD bFGF fragment) is used. The protein sequence of 18kD bFGF is shown as SEQ ID NO: 2. In accordance with one embodiment, the coding sequence of bFGF is operably linked to an inducible promoter selected from the group consisting of promoters that are induced by the following compounds: tetracycline and its analogs (such as doxycycline) (M. Gossen et al., 1995, Science 268:1766-1769); rapamycin (V.M. Rivera et al., 1996, Nature Med 2:1028-1032), estradiol analogs (J Whelan and N Miller, 1996, J Steroid Biochem Mol Biol 58:3-12), ecdysone and analogs (such as muristerone A and ponasterone A) (D No et al., 1996, Proc Natl Acad Sci USA 93:3346-3351); or progesterone antigonists (such as RU486 and mifepristone) (Y Wang et al, 1994, Proc Natl Acad Sci USA 91 :8180-8184). In one preferred embodiment the inducible promoter is selected from a tetracycline, ecdysone or estradiol inducible promoter.

The nucleic acid sequence comprising the growth factor operably linked to an inducible promoter is delivered to the site of the wound in a manner that allows for controlled expression of the growth factor. In particular, the DNA construct is delivered in accordance with one of three general routes: 1) direct administration of naked DNA, 2) administration of the nucleic acid construct packaged or linked to a non-cellular delivery vehicle or 3) administration of cells that have been transfected with the nucleic acid construct. The first two methods for delivering the gene product to the wound site rely on the ability of endogenous cells present at the wound site to take up the nucleic acid construct and express the gene product.

In accordance with one embodiment of the present invention a wound healing composition comprises a nucleic acid construct packaged in a delivery vehicle. The delivery vehicle can be selected from polymeric matrixes such as hydrogels, liposomes, cationic transfection reagents, collagen matrixes, gauze (impregnated with the wound healing composition) or biological vectors including viral and retroviral vectors such as adenovirus, AAV, and lentivirus. The nucleic acid sequences are taken up by cells adjacent to the wound and the genes are expressed, only in the presence of the appropriate inducer, to provide a controlled supply of growth factor to the wound site. For example the DNA construct may comprise one or more growth factors selected from the group consisting of FGF, PDGF, EGF and TGF-β wherein the growth factor is operably linked to an inducible promoter. In one embodiment the growth factor is bFGF. In accordance with one embodiment the nucleic acid sequence packaged into the delivery vehicle encodes several growth factors and in one embodiment the sequence also encode for a cytokine such as Interleukin 4. In this embodiment each of the growth factors and cytokines are linked to different inducible promoters. In this manner, the wound healing composition can be administered to a wound and the therapeutic regiment can be modified simply by altering the type and amount of inducers used to stimulate expression from the recombinant genes. Advantageously, in this embodiment a single wound healing composition can be used to treat many different types of wounds simply by altering the inducers used to stimulate expression of the growth factor genes.

In accordance with another embodiment, the wound healing composition comprises fibroblasts transformed with a nucleic acid sequence, wherein the nucleic acid sequence encodes a growth factor, selected from the group consisting of FGF, PDGF, EGF and TGF-β, and/or a cytokine. A secretory sequence is also preferably operably linked to the sequence encoding the growth factor. In one embodiment the secretory sequence is linked to a low molecular weight bFGF, which should act in an autocrine, as well as paracrine fashion, to promote tissue regeneration in large soft tissue defects. Any of the eukaryotic peptide secretory sequences known to those skilled in the art can be used in the present invention. One example is the preprotrypsin sequence as shown as SEQ ID NO: 3. Other secretory sequences suitable for use in the present invention include, but are not limited to, the V-J2-C region of the Ig kappa chain or the signal sequences for the E. coli malE gene or interleukins. In one preferred embodiment the preprotrypsin secretory sequence is linked to the 5' end of the 18kD bFGF sequence and inserted into the pFlag expression vector to enhance the secretion of the expressed FGF fusion protein from the transformed fibroblast cells. Use of the Flag expression vector along with the preprotrypsin signal sequence is described by R. G. Chubert and Bill L Brizzard, 1996, BioTechniques 20:136-141. The vector constructs/delivery vehicle or genetically modified cells are injected locally around the wound to induce gene expression and tissue healing. In accordance with one embodiment an effective amount of the wound healing composition is injected subcutaneously at one or more sites about the periphery of the wound. The number of injections and the amount of material injected will be varied in accordance with the size and severity of the wound and the composition of the wound healing composition.

For example, autologous fibroblast cells can be obtained from the patient and transformed in vitro with a nucleic acid construct comprising a growth factor operably linked to an inducible promoter. The transgenic fibroblasts can then be injected into the cells bordering the wound site, wherein the injected cells secrete the growth factor upon stimulation by the inducer agent. Secretion of the growth factors enhances cellular proliferation, angiogenesis and scar formation.

In an alternative embodiment the nucleic acid sequence construct (comprising a growth factor operably linked to an inducible promoter) is encapsulated in an adenovirus or other viral vector. The viral delivery vehicle is then place in contact with the cell bordering the wound site to transfect those cells with the nucleic acid construct. For example, a pharmaceutically acceptable composition comprising the viral delivery vehicle can be injected into the tissue surrounding the wound. The viral delivery vehicle introduces the nucleic acid construct into fibroblasts and other cells involved in the repair process allowing for the controlled expression of the growth factor. Appropriate expression of the growth factor induces beneficial cellular proliferation, angiogenesis and scar formation. The nucleic acid construct may further comprise a protein secretory signal sequence operably linked to the growth factor coding sequence to enhance the secretion of the growth factor by the transformed cells.

In accordance with the present invention, the expression of the growth factor is regulated by an inducible promoter or tissue specific promoter to control or limit transgene expression. The use of an inducible promoter to control the expression of the growth factor is important in order to decrease the possibility of tumor formation. Typically the expression of the growth factor will be continued until complete wound healing is accomplished, however the amount of growth factor produced can be altered as tissue repair is completed. For example, to initiate wound healing, a higher local concentration of growth factor may be desirable at the early stages of wound healing relative to later stages of the healing process. In accordance with one embodiment, the amount of growth factor expressed is controlled by regulating the amount of inducer compound that contacts the wound. In preferred embodiments the inducer is administered orally, however other routes of administration, such as topical or intravenous administration are also contemplated. One preferred inducible system for expressing the growth factor is the Tet-On system that is commercially available from Clonetech Laboratories Inc., 1020 East Meadows Circle, Palo Alto, C A 94303. Another inducible system that can be used in accordance with the present invention is the Ecdysone-Inducible Mammalian Expression System (T-Rex system), commercially available from Invitrogen Corporation, 1600 Faraday Ave, Carlsbad, CA 92008.

It would also be possible to utilize two or more different inducible expression systems with two or more different transgenes to optimize tissue healing. For example, in addition to FGF, other proteins such as TGF-β, VEGF, EGF, PDGF, or other cytokines (alone or in combination) can be administered in a similar fashion, with inducible or tissue specific promoters. In accordance with one embodiment a wound healing composition comprises a nucleic acid sequence encoding bFGF that is operably linked to a first inducible promoter and a nucleic acid sequence encoding a second growth factor or a cytokine that is operably linked to a second inducible promoter. Preferably, expression from the first and second inducible promoters is regulated by different inducible agents.

In accordance with one embodiment of the present invention a method is provided for enhancing the repair of wounds. The method comprises the steps of contacting the wound site with the wound healing composition of the present invention and inducing the expression of the recombinant growth factor gene.

Thus the present invention provides a novel method for regulating localized levels of growth factors and/or cytokines. In accordance with one embodiment the method comprising the steps of contacting a target site with a composition comprising a nucleic acid sequence that encodes a growth factor selected from the group consisting of Fibroblast Growth Factor, Platelet Derived Growth Factor, Epidermal Growth Factor and Tissue Growth Factor β, wherein the growth factor is operably linked to an inducible promoter. The expression of the growth factor is expressed in low levels or not at all until stimulated by the presence of an inducer agent. By regulating the concentration of the inducer agent, the amount of expression of the recombinant growth factor gene can be regulated thus regulating the local concentration of the growth factor.

In one preferred embodiment a method for enhancing the repair of wounds comprises the steps of contacting cells at the periphery of said wound with a composition that comprises a nucleic acid construct, wherein the nucleic acid construct encodes the bFGF coding sequence operably linked to an inducible promoter. The cells at the periphery of the wound are contacted in accordance with one embodiment by injecting the wound healing composition subcutaneously at the wound site. Alternatively, or in addition to the subcutaneous injection, a topically applied composition can be used to place the present wound healing compositions in contact with the cells at the periphery of said wound. For example the wound healing composition of the present invention can be formulated as a wound dressing. The wound healing compositions of the present invention can be formulated with standard pharmaceutically acceptable carriers or excipients using standard techniques known to those skilled in the art.

Example 1

Inducible Systems Suitable for use with the Present Invention Tet-Off™™ & Tet-On^TM™ Gene Expression System

Regulated, high-level gene expression systems were first described by Bujard, Gossen, and colleagues (Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551 and Gossen, M., et al. (1995) Science 268:1766-1769). Tet- Off and Tet-On Gene Expression Systems allow high-level, regulated gene expression in response to varying concentrations of tetracycline (Tc) or Tc derivatives such as doxycycline (Dox). In the Tet-Off System, gene expression is turned on in the absence of Tc or Dox. In contrast, gene expression is activated in the Tet-On System in the presence of Dox.

The Tet Expression Systems are based on two regulatory elements derived from the tetracycline-resistance operon of the E. coli TnlO transposon: the tetracycline repressor protein (TetR) and the tetracycline operator sequence (tetO) to which TetR binds. The gene to be expressed (gene X) is cloned into the pTRE2 "response" plasmid, which contains the _hCMV promoter upstream of a multiple cloning site (MCS). P_hCMv_*-_\ ^{1S a} compound promoter consisting of the tetracycline- responsive element (TRE), which contains seven copies of tetO, and the minimal immediate early promoter of cytomegalo virus (-P_mιnCMv)-

The second key component of the system is a "regulator" plasmid which expresses a hybrid protein known as the Tc-controlled transactivator (tTA). tTA is encoded by pTet-Off and is a fusion of the wild-type TetR to the VP16 activation domain (AD) of herpes simplex virus. tTA binds the tetO sequences which brings the VP16 activation domain into close proximity with the P_hCw_*-ι C³¹¹⁰" thereby activates transcription) in the absence of Tc. Thus, as Tc is added, transcription is turned off in a dose-dependent manner. The Tet-On System is based on the "reverse" TetR (rTetR), which differs from the wild-type TetR by four amino acid changes. When fused to the VP16 AD, rTetR creates a "reverse" tTA (rtTA) that activates transcription in the presence of Dox. The Ecdysone-Inducible Mammalian Expression System

Another inducible system that can be used in accordance with the present invention is the Ecdysone-Inducible Mammalian Expression System. The system is designed for tightly-regulated expression of a gene of interest in mammalian cells and is based on a unique insect mechanism. There is almost no detectable basal expression and greater than 200-fold inducibility can be achieved in mammalian cells. In the Ecdysone-Inducible Mammalian Expression System, both subunits of a functional ecdysone receptor from Drosophila are constitutively expressed from the regulator vector pVgRXR. The ecdysone-responsive promoter (p-Δ-HSP)— which ultimately drives expression of the gene of interest—is located on a second inducible expression vector. Mammalian cells are cotransfected with an inducible expression vector containing the gene of interest and pVgRXR. In the presence of the inducer (ponasterone A or muristerone A) the functional ecdysone receptor binds upstream of the ecdysone responsive promoter and activates expression of the gene of interest.

Example 2

Inducible Expression of FGF

To determine if bFGF could be secreted from fibroblast cells in an inducible manner, rabbit fibroblast cells (HIG cells) were transformed with a bFGF gene construct and cultured in the presence and absence of the inducer. The construct comprises a tetracycline inducible regulatory element upstream from the sequence encoding the bFGF protein. The bFGF sequence encodes a truncated version of bFGF (the 18kD version) with a secretory peptide (preprotrypsin) linked to the amino terminus of the 18kD bFGF (SEQ ID NO:). The transformed HIG cells were cultured in standard eukaryotic tissue media under standard culture conditions. The cells were divided into two groups of approximately 6.4 x 10³ cells. The first group of cells served as a control whereas the second group was administered lOug/ml doxycycline, an inducer of the tetracycline inducible regulatory element. After a preselected time of incubating the cells, the cells were removed and the supernatant was screened, using an ELISA assay, for the presence of bFGF. The results were as follows:

In the supernatant isolated from cells exposed to lOug/ml of doxycycline the concentration of bFGF was determined to be 103.9 pg/ml. In the supernatant isolated from cells from the control group (no doxycycline) the concentration of bFGF was determined to be -15.24 pg/ml. Therefore the secretion of bFGF can be tightly regulated by the administration of an inducer of the tetracycline regulatory element.

Example 3

Use of Transformed Fibroblast to Enhance Wound Repair

Rats were used in this study since they are the easiest and smallest animal which is still large enough to develop the pressure ulcer model. More particularly, nude rats were used first since human bFGF (basic fibroblast growth factor) and TIMP (tissue inhibitor of metalloproteinase) will be released from the transfected human fibroblasts and may elicit an immune response to the foreign protein and cells in rodents. Anesthesia and analgesics were be used before and after surgery. Animals were anesthetized and aseptically prepped for surgery. Metal or magnetic plates were implanted on the dorsum of animals and animals were recovered from anesthesia before being returned to vivarium. One month after implantation a magnetic disc was attached over the metal plates for 6 hours a day until the pressure ulcer developed (6-7 days). The animals were observed for any problem or distress. Analgesic is usually not needed, but was used when necessary

(Buprenorphine 0. lmg/kg sg bid 24hrs). Quality of the wound was monitored before each magnetic disc application and after transplantation of fibroblasts. The animals were prophylactically treated with tetracycline water until euthanasia.

After application of last pressure session and while under anesthesia, therapeutic gene-transfected fibroblasts (i.e. fibroblasts containing gene encoding

FGF) were transplanted to each wound. Recovery from the anesthesia was completed before the animals are returned to the vivarium. On days 1, 3, 5, 7, 9, 11, 13, 15, 30, and 60 wound quality was monitored; animal anesthetized, ulcer biopsied for histology and animal euthanized, and tissue homogenized to determine the concentration of gene product. Preliminary Data DAY 1 - Insertion of subcutaneous magnet DAY 30 - Begin pressure treatments DAY 37- Pressure ulcer developed

Injections Results

1) FGF secreting fibroblasts N=3 Complete healing Day 8

2) B-gal transduced fibroblasts N=3 Incomplete healing Day 15

3) Control- No treatment N=3 Incomplete healing Day 15

1) Secretory sequence - FGF DNA N= 1 Complete healing Day 7

2) No treatment - Control N= 1 Incomplete healing Day 7

Example 4 Prevention of pressure ulcer

A metal plate will be implanted into the dorsum of each animal as describe in Example 3. After 1 month of implantation, the animals will be anesthetized and DNA plasmids encoding FGF under the control of an inducible promoter will be injected into the muscle of one implantation site. The animals will be recovered from the anesthesia before being returned to the vivarium. Several days later, when injected plasmid is activated (by the presence of the inducer), the animals will go through the pressure ulcer procedure.

At staggered intervals until development of pressure ulcer, the animals will be anesthetized and punch biopsies will be performed on both control and DNA injected ulcer sites, followed by euthanasia of the animals.

Claims

Claims:

1. A wound healing composition comprising a delivery vehicle; and a nucleic acid sequence that encodes a growth factor selected from the group consisting of Fibroblast Growth Factor, Platelet Derived Growth Factor, Epidermal Growth Factor and Tissue Growth Factor β, said growth factor being operably linked to an inducible promoter, and said nucleic acid being linked to said delivery vehicle.

2. The wound healing composition of claim 1 wherein the delivery vehicle is selected from the group consisting of adenovirus, AAV, lentivirus, liposome, SV40 and cationic transfection agent.

3. The wound healing composition of claim 1 wherein the growth factor is basic Fibroblast Growth Factor.

4. The wound healing composition of claim 1 wherein the nucleic acid sequence encoding basic Fibroblast Growth Factor consists of SEQ ID NO: 1.

5. A method for regulating localized levels of growth factors, said method comprising the steps of contacting a target site with a composition comprising a nucleic acid sequence that encodes a growth factor selected from the group consisting of Fibroblast Growth Factor, Platelet Derived Growth Factor, Epidermal Growth Factor and Tissue Growth Factor β, said growth factor being operably linked to an inducible promoter; and adding an inducer that enhances expression of the growth factor.

6. The method of claim 5 wherein the growth factor is basic Fibroblast Growth Factor.

7. The method of claim 6 wherein the composition further comprises fibroblast cells that have been transformed with said nucleic acid sequence.

8. The method of claim 7 wherein the nucleic acid sequence encoding basic Fibroblast Growth Factor further comprises a protein secretory signal sequence operably linked to sequence encoding basic Fibroblast Growth Factor.

9. The method of claim 6 wherein the composition further comprises a delivery vehicle linked to the nucleic acid sequence.

10. The method of claim 9 wherein the delivery vehicle is an adeno viral or AAV vector.

11. The method of claim 10 wherein the nucleic acid sequence encoding basic Fibroblast Growth Factor consists of SEQ ID NO: 1.

12. A composition for enhancing the repair of wounds, said composition comprising a nucleic acid sequence comprising SEQ ID NO: 1, wherein SEQ ID NO: 1 is operably linked to an inducible promoter.

13. The composition of claim 12 wherein the nucleic acid sequence further comprises a peptide secretory sequence operably linked to SEQ ID NO: 1.

14. The composition of claim 12 wherein the wound to be healed is a pressure ulcer.

15. A wound healing composition comprising a nucleic acid sequence encoding basic Fibroblast Growth Factor wherein the basic Fibroblast Growth Factor is operably linked to a first inducible promoter; a nucleic acid sequence encoding a second growth factor or a cytokine wherein the second growth factor or cytokine is operably linked to a second inducible promoter, wherein expression of the sequences operably linked to the first and second inducible promoters is regulated by different inducible agents.