JP2001511462A - Graft treatment - Google Patents

Abstract

  (57) [Summary] The present invention provides a method of treating a graft, comprising introducing a nucleic acid encoding an angiogenic substance into the graft cells. The graft may be treated ex vivo and then transplanted into a donor or may be treated post-transplant. The graft may be an autologous, allogeneic, xenogeneic, or tissue engineered graft ("bioartificial" organ). Nucleic acids may be introduced into cultured cells used to make tissue engineering implants. The expression of angiogenic substances by the graft cells promotes the growth of new blood vessels (angiogenesis) that supplies the graft with blood and thereby increases the likelihood of graft survival.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Successful transplantation of an allograft in a host can be attributed to antigens on the transplanted tissue that the recipient may recognize as foreign and cause rejection, cells that mediate rejection in the recipient's immune system, and foreign It depends on factors such as the response that alters either the presentation of the antigen or the cellular response.

[0002] To minimize tissue rejection, the use of immunosuppressants can suppress the immune response to transplanted tissues, but by their nature, immunosuppressive therapy is systemic. Thus, immunosuppressants tend to suppress the immune response, thereby reducing the transplant patient's ability to fight infection.

[0003] In view of these complications, transplant immunologists have sought ways to suppress the immune response in an antigen-specific manner (responding to only the donor alloantigen).
For example, in vitro treatment with cortisone, thalidomide, or urethane before implanting skin grafts in laboratory animals doubled the skin graft survival time. The amount of drug applied topically to the skin was less than that required for a systemic injection to achieve an equivalent effect. In another test, donor skin was treated in vitro with streptokinase / streptodolase, or recipient RNA and DNA preparations. Furthermore, it was found that treating the transplanted tissue with a glutaraldehyde solution before transplantation reduced its antigenicity. See U.S. Pat. No. 4,120,649.

[0004] TGF-beta has been shown to suppress human interferon gamma-induced expression of class II histocompatibility antigens on human cells and inhibit constitutive expression of class II antigen messages in cells . It has been proposed to use recombinant TGF-beta as an immunosuppressant for graft treatment prior to implantation. US Patent No. 51
See 35915.

[0005] In addition to immune rejection, many grafts, especially skin grafts, do not survive due to an inadequate blood supply. This is especially common in patients who develop ulcers due to limb ischemia.

[0006] It is desirable to develop a method of prolonging graft survival that minimizes the toxicity and other adverse effects caused by using large amounts of immunosuppressive drugs during transplant surgery.

[0007] Further, in the treatment of skin ulcers in patients with limb ischemia, it is desirable to develop methods for prolonging graft survival and wound healing.

SUMMARY OF THE INVENTION [0008] The present invention provides a method of treating a graft, comprising introducing a nucleic acid encoding an angiogenic substance into the graft cells. The graft may be treated ex vivo and then transplanted into a donor, or may be treated post-transplant. The graft may be an autologous, allogeneic, xenogeneic, or tissue-engineered graft ("bioartificial" organ). Nucleic acids may be introduced into cultured cells used to make tissue engineering implants. The expression of angiogenic substances by the graft cells promotes the growth of new blood vessels (angiogenesis) that supplies the graft with blood and thereby increases the likelihood of graft survival.

Another aspect of the present invention is disclosed below.

DETAILED DESCRIPTION OF THE INVENTION [0010] As used herein, the term "graft" refers to biological material from a donor for transplantation into a recipient. Implants include tissues and organs that benefit from angiogenesis. Organs include, for example, skin, heart, liver, spleen, pancreas, thyroid lobule, lung, kidney, tubular organs (eg, intestine, blood vessels, or esophagus), and the like. Tubular organs can be used to replace damaged sites in the esophagus, blood vessels, or bile ducts. Skin grafts can be used not only for ischemic skin ulcers and burns, but also as dressings to the damaged intestine or to close certain defects such as diaphragmatic hernias. The graft is provided from any organism, preferably from either a cadaver or a living donor, including mammals, including humans. Alternatively, the implant may be a tissue engineered implant made from a combination of cultured cells and skeletal material. An example of such a tissue engineering implant is Appligraf®. Appligraf® consists of a type I collagen gel inoculated with allogeneic fibroblasts covered with a confluent surface of allogeneic keratinocytes.

As used herein, the term “host” refers to any compatible transplant recipient. By the term "compatibility" is meant that the host accepts the donated graft. Preferably the host is a mammal, more preferably a human. If both the donor and the host of the graft are human, it is preferred that they are HLA class II antigen matched to improve histocompatibility.

As used herein, the term “donor” refers to a dead or living species from which an implant is obtained. Preferably, the donor is a mammal. It is preferred that human donors be clinically normal and have the same relative ABO blood type as donor donors, as crossing the major blood group barrier may interfere with allograft engraftment. However, it is possible, for example, to transplant a type O donor kidney into a type A, type B or type AB recipient.

The terms “graft” and “implanted tissue” are used interchangeably and refer to a tissue or cell (heterologous or allogeneic) that is introduced into the body of a host and replaces the structure or function of the endogenous tissue.

[0014] The term "angiogen" refers to any protein, polypeptide, mutein, or portion that can directly or indirectly promote the formation of new blood vessels. Folkman et al., Science, 235: 442-447 (1987). Such proteins include:
For example, acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2)
, FGF-4, FGF-5, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming growth factors α and β (TGF-α and TGF-β), platelet-derived endothelial growth factor (PD-ECG
F), platelet-derived growth factor (PDGF), tumor necrosis factor α (TNF-α), hepatocyte growth factor (HGF, scattering factor), insulin-like growth factor (IGF), IL-8, proliferin,
Angiogenin, fibrin fragment E, angiotropin, erythropoietin, colony stimulating factor (CSF), macrophage-CSF (M-CSF), granulocyte /
Includes macrophage CSF (GM-CSF) and nitric oxide synthase (NOS). VE
GFs include various forms of VEGF, such as VEGF ₁₂₁ , VEGF ₁₄₅ , VEGF ₁₆₅ and VEGF ₁₈₉ . Klagsbrun et al., Annu. Rev. Physiol., 53: 217-239 (1991); Folkman et al., J. B.
iol. Chem., 267: 10931-10934 (1992) and Symes et al., Current Opinion in Lipi.
dology, 5: 305-312 (1994).

Preferably, the angiogenic protein contains a secretory signal sequence that promotes secretion of the protein. Angiogenic proteins having a native signal sequence, such as VEGF, are preferred. Angiogenic proteins without a native signal sequence, such as bEGE, can be modified to include such sequences using conventional genetic engineering techniques. Nable et al., Nature, 3
62: 844 (1993).

The angiogenic effect of any given protein, peptide or mutein can be determined, for example, by the rabbit corneal pocket assay (Gaudric et al., Ophthalmic. Res. 24: 181-8 (19
92)) and chicken chorioallantoic membrane (CAM) assay (Peek et al., Exp. Pathol. 34: 35-40).
(1988)).

[0017] The nucleotide sequences of a number of angiogenic proteins are, for example, GenBank, EMBL and Swi
It can be easily checked from many computer databases such as ss-Prot. Using this information, a DNA segment encoding the desired one can be chemically synthesized, or such a DNA segment can be obtained using methods conventional in the art, for example, PCR amplification.

To simplify manipulation and handling of the nucleic acid encoding the protein, the nucleic acid is preferably inserted into a cassette where it is linked to a promoter. The promoter must be able to drive protein expression in the desired target tissue cells. Selection of an appropriate promoter can be easily performed. Preferably, a high expression promoter is used. An example of a suitable promoter is the 763 base pair cytomegalovirus (CMV) promoter. Rous sarcoma virus (RSV) (Davis et al., H
um Gene Ther 4: 151 (1993)) and the MMT promoter can also be used. Certain proteins can be expressed using their native promoter. Other factors that can enhance expression may also be included, such as enhancers or tat genes and systems that cause high levels of expression such as tar factors. This cassette can then be inserted into a vector, for example, a plasmid vector such as pUC118, pBR322, or other well-known plasmid vectors containing, for example, an E. coli origin of replication. Sambrook et al., Molecular Cloning: An Experimental Guide to Molecular Clonin
g: See A Laboratory Manual, Cold Spring Harbor Laboratory press, (1989). The plasmid vector may include a selectable marker, such as the β-lactamase gene for ampicillin resistance, provided that the marker polypeptide does not deleteriously affect the metabolism of the organism being treated. The cassette can also be linked to a nucleic acid binding moiety in a synthetic delivery system such as the system disclosed in WO 95/22618.

In certain situations, it may be desirable to use nucleic acids encoding two or more different proteins to optimize therapeutic outcome. For example, DNAs encoding two angiogenic proteins, for example, VEGF and bFGF, can be used, resulting in an improvement over using bFGF alone. Alternatively, angiogenic factors can be combined with other genes or the gene products they encode to induce angiogenesis and at the same time enhance the activity of target cells, including, for example, nitric oxide synthase, L-arginine ,
Includes fibronectin, urokinase, plasminogen activator and heparin.

The term “effective amount” means an amount of nucleic acid sufficient to produce a sufficient level of angiogenic protein, ie, a level that can induce angiogenesis. Thus, an important aspect is the level of the expressed protein. Thus, multiple transcripts can be used or the gene can be under the control of a promoter, resulting in a high level of expression. In another embodiment, the gene may be controlled by factors that result in very high levels of expression, such as tat and the corresponding tar factor.

Usually, the nucleic acid encoding the angiogenic agent is a physiologically acceptable carrier at room temperature, at a suitable pH and at the desired purity, ie, a carrier that is not toxic to the recipient at the applied doses and concentrations. And by mixing.

The nucleic acid is introduced into the graft cells by any method that results in the cell taking up and expressing the nucleic acid. Transduction can be by standard methods, such as infection, transfection, transduction or transformation. Examples of modes of gene transfer include, for example, naked DNA, Ca ₃ (PO ₄ ) ₂ precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, viral vectors, adjuvant-assisted DNA, catheters, gene guns and so on. Vectors include chemical conjugates, such as those described in WO 93/04701, having a targeting moiety (eg, a ligand for a cell surface receptor) and a nucleic acid binding moiety (eg, polylysine), a viral vector. (Eg, a DNA or RNA viral vector), including a targeting moiety (eg, an antibody specific for the target cell) and a nucleic acid binding moiety (eg, protamine)
It includes fusion proteins, plasmids, phages, etc. as described in PCT / US95 / 02140 (WO 95/22618). Vectors can be chromosomal, non-chromosomal,
Or it may be by synthesis.

[0023] Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpes simplex I virus (HSV) vectors [AI Geller et al., J. Neurochem, 64: 487 (1995); F. Lim et al.
NA Cloning: Mammalian DNA Cloning: Mammalian Systems, D. Glover (O
xford Univ. Press, Oxford, UK (1995); AI Geller et al., Proc.
Natl. Sci .: USA: 90 7603 (1993); AI Geller et al., Proc. Natl. Sci. USA.
: 87: 1149 (1990)], adenovirus vectors [LeGal LaSalle et al., Science, 259: 988 (1993); Davidson et al., Nat. Genet., 3: 2.
19 (1993); Yang et al., J. Virol., 69: 2004 (1995)] and adeno-associated virus vectors [Kaplitt, MG et al., Nat. Genet., 8: 148 (1994)].

Poxvirus vectors introduce a gene into the cytoplasm of a cell. Avipoxvirus vectors only express nucleic acids for short periods of time. For introducing nucleic acids into nerve cells, adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors are preferred. Expression with adenovirus vectors (about 2 months) is shorter than adeno-associated virus (about 4 months), but adeno-associated virus vectors are shorter than HSV vectors. The type of vector selected will depend on the target cell and the condition being treated.

Gene guns include those disclosed in US Pat. Nos. 5,100,792 and 5,37,115, and PCT Publication No. WO 91/07487.

If desired, the nucleic acids may be used with microdelivery vehicles such as cationic liposomes and adenovirus vectors. For a review of liposome preparation, targeting, and content delivery methods, see Mannino and Gould-Fogerite, B
See ioTechniques, 6: 682 (1988). Felgner and Holm, Bethesda Res.Lab.Focus, 11 (2): 21 (1989) and Maurer, RA, Bethesda Res.Lab.Focus.
, 11 (2): 25 (1989).

[0027] Replication-defective recombinant adenovirus vectors can be produced according to well-known methods. Quantin et al., Proc. Natl. Acad. Sci. USA, 89: 2581-2584 (1992).
Stratford-Perricadet et al., J. Clin. Invest., 90: 626-630 (1992); and Ros.
See enfeld et al., Cell, 68: 143-155 (1992).

To deliver the nucleic acid to the skin graft, the graft may be immersed in the nucleic acid composition for a sufficient time to incorporate the nucleic acid.

For use in tissue engineering implants, the cells used to make the implant are transfected with a nucleic acid encoding an angiogenic agent. Preferably, the cells are transfected before making the graft. For example, the registered trademark Apligraph (Organogenes
In tissue-engineered grafts, such as synthetic skin equivalents (e.g., Canton, Mass.), keratinocytes used to make the grafts are transfected in culture with a vector containing DNA encoding angiogenic substances. be able to.

[0030] Nucleic acids may be introduced by direct injection into the graft, before or after transplantation.

[0031] The nucleic acids can be applied topically prior to implantation, eg, on a skin graft. In such a case, it is preferable to use a viscous solution such as a gel rather than a non-viscous solution. This is accomplished, for example, by mixing the nucleic acid solution with a gelling agent such as a polysaccharide, preferably, for example, hyaluronic acid, starch, and water-soluble polysaccharides such as cellulose derivatives, for example, methylcellulose, hydroxyethylcellulose, and carboxymethylcellulose. . The polysaccharide is generally present in the gel formulation in a gel weight ratio of 1-90%, more preferably 1-20%. Examples of other polysaccharides and polysaccharide solubility assays suitable for this purpose are described in EP 267015 published May 11, 1988, the disclosure of which is incorporated herein by reference. I do.

In certain situations, nucleic acids are introduced by contacting the graft with the nucleic acids in a suitable composition. The contacting is suitably incubating the organ with or perfusing the composition with the composition, or applying the composition to one or more surfaces of the graft for a sufficient time to allow the cells of the graft to take up the nucleic acid. . The treatment is generally performed for at least 1 minute, preferably 1 minute to 72 hours, more preferably 2 minutes to 24 hours, depending on factors such as the nucleic acid concentration in the formulation, the implant to be treated, and the type of formulation. Perfusion is achieved in any suitable manner. For example, as described in DD213134 published on September 5, 1984, an organ can be perfused by a device that includes a pressure regulator and a drain port between the pump and the organ and provides a constant perfusion pressure. . Alternatively, as described in European Patent No. 125847 published on November 21, 1984, the organ is placed in a high pressure chamber through a sealed door, and the perfusate is pumped from a reservoir to the high pressure chamber while spent perfusion is performed. The liquid is returned to the storage tank by the valve.

Prior to transplantation, the host may be subjected to pretransplantation measures that are beneficial to the particular transplant recipient.

The transplantation procedure itself depends on the disease to be treated, the condition of the patient, and the like. The physician will identify the appropriate course of treatment for any given case. During the postoperative danger period (the first three months), the grafts are optionally systematically monitored using appropriate methods. After transplantation, immunosuppressive therapy may be used as needed to ensure graft engraftment.

[Brief description of the drawings]

FIG. 1 is a view showing a photograph of a mouse model in which a full-thickness skin wound was created on the dorsal outer skin of the upper spine.

FIG. 2 is a diagram showing macroscopic findings of a graft placed on the wound shown in FIG. 1. In this case, explants were generated by transfecting keratinocytes in culture with an adenovirus construct encoding beta-galactosidase. After incorporating keratinocytes into the skin used to perform the transplant,
The grafts were allowed to survive for 7 days, at which time the grafts were removed with normal skin borders and then stained with X-Gal to identify staining related to beta-galactosidase expression in keratinocytes of the grafts .

FIGS. 3A-3C show micrographs of control grafts (3A) prepared from keratinocytes not transfected with beta-galactosidase. FIGS. 3B and 3C show grafts prepared from keratinocytes transduced (driven by the cytomegalovirus promoter) with an adenovirus beta-galactosidase construct at a multiplicity of infection of 37 (3B) and 150 (3C). It is. The deep staining identifies keratinocytes actively expressing the lacZ transgene encoding beta-galactosidase.

Claims (4)

[Claims] 1. A method of treating a graft, comprising introducing an effective amount of a nucleic acid encoding an angiogenic substance into the graft cells. 2. The method of claim 1, wherein the nucleic acid is contacted prior to transplanting the graft into a compatible host. 3. The method of claim 1, wherein the implant is tissue. 4. The method of claim 1, wherein the implant is skin.