DE4219626A1 - Incorporating therapeutic gene via vector into body cells - in=vivo or in vitro, for subsequent expression and secretion of active protein, partic. for treating degenerative diseases of spine and nerves - Google Patents

Incorporating therapeutic gene via vector into body cells - in=vivo or in vitro, for subsequent expression and secretion of active protein, partic. for treating degenerative diseases of spine and nerves

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DE4219626A1
DE4219626A1 DE19924219626 DE4219626A DE4219626A1 DE 4219626 A1 DE4219626 A1 DE 4219626A1 DE 19924219626 DE19924219626 DE 19924219626 DE 4219626 A DE4219626 A DE 4219626A DE 4219626 A1 DE4219626 A1 DE 4219626A1
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Peter Wehling
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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

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Abstract

Method comprises incorporation of a therapeutic gene, by means of vectors, into body cells with subsequent expression, by the genetically modified cells, of therapeutic protein (I) and secretion of (I) into the extracellular environment. Pref. cells are nerve cells, immune competent cells, mesenchymal and ectodermal cells, esp. peripheral nerve cells, macrophages, lymphocytes, fibroblasts and chondrocytes. Pref. (I) have antiinflammatory, analgesic, regenerative, immunostimulating, hypotensive, anti-degenerative or antiarthrotic activities. The vector is pref. a retro-, adeno-, adeno-associated or herpes-virus, or a liposome, and may be injected directly, in vivo. Alternatively cells are removed, those cells capable of division selected and the gene introduced in vitro. The modified cells are returned to the donor. In this case the transfected cells may be indentified by co-transfection with a marker. Pref. IL-1 antagonists are IL-1 receptor antagonist and IL-1 receptor. USE/ADVANTAGE - The method is esp. used to express cytokines (or their inhibitors); opiates; prostaglandins (sic) and their inhibitors; esp. inhibitors of interleukin-1. Esp. is is used to treat degenerative diseases of the spinal column and nerves. Gene transfer should elminate the need for large, and frequent, injections of exogenous proteins which have only short half lives in tissue.

Description

1. Einleitung1 Introduction

Degenerative Erkrankungen des Bewegungsappa­ rates insbesondere der Wirbelsäule stellen den häufigsten Erkrankungstyp des älteren Menschen dar. Nach Untersuchungen des "National Center for Health Statistics" leiden annähernd 500 von 1000 Personen über 65 Jahren an degenerativen Erkran­ kungen. Die Zahlen dürften für die Bundesrepublik ähnlich hoch liegen.Degenerative diseases of the musculoskeletal system councils especially the spine represent the most common type of disease in the elderly According to investigations by the "National Center for Health Statistics "affects approximately 500 out of 1000 People over 65 years of age with degenerative disease kungen. The numbers are likely for the Federal Republic are similarly high.

Bandscheibe und Wirbelsäulengelenke sind Orga­ ne, die mit Medikamenten schwierig zu erreichen sind. Intravenöse und orale Verabreichungen von Medikamenten mit potentiell analgetischer und ar­ throsehemmender Wirkung erreichen nur bedingt den Bandscheibenraum bzw. den Innenraum ar­ throtisch veränderter kleinen Wirbelgelenke, da die entsprechenden Strukturen verminderten Anschluß an das vaskuläre System haben. Vermindert wer­ den die Gewebespiegel therapeutischer Substan­ zen in der Bandscheibe und den kleinen Wirbelge­ lenke außerdem durch die passive Diffusion von den Kapillaren in die Bandscheibe bzw. den Gelen­ kinnenraum. Dabei gilt, daß je größer das verab­ reichte Molekül ist, um so niedriger die Diffusion in diese Zielgebiete ist. Der Zugang von großen thera­ peutisch wirksamen Molekülen (z. B. Proteinen) in diese Areale ist damit wesentlich erschwert.The intervertebral disc and spinal joints are org ne that difficult to achieve with medication are. Intravenous and oral administrations of Drugs with potentially analgesic and ar Achieve throses only limited the intervertebral disc space or the interior ar throtically modified small vertebral joints, because the corresponding structures reduced connection to the vascular system. Diminishes who the tissue level of therapeutic substance zen in the intervertebral disc and the small vertebrae also steer through the passive diffusion of the capillaries in the intervertebral disc or the gels interior. It applies that the larger the rich molecule is, the lower the diffusion in this is target areas. The access of great thera peutically active molecules (e.g. proteins) in these areas are therefore considerably more difficult.

Obwohl intraartikuläre und intradiskale Injektionen einen direkten Zugang von Medikamenten ermögli­ chen und damit die genannten Probleme umgehen, haben therapeutisch interessante Substanzen (z. B. IL-1 Antagonisten) eine kurze Halbwertzeit im Ge­ webe. Wegen der Chronizität sind viele Injektionen erforderlich. Wiederholte Injektionen bergen das Risiko der bakteriellen Infektion.Although intra-articular and intradiscal injections direct access to medication and thus deal with the problems mentioned, have therapeutically interesting substances (e.g. IL-1 antagonists) have a short half-life in Ge weave. Because of the chronicity there are many injections required. Repeated injections hide that Risk of bacterial infection.

Bisher ist es daher üblich, systemisch mit hohen Gewebespiegeln zu therapieren, um so eine effekti­ ve therapeutische Dosis in der Zielstruktur (Band­ scheibe u. Wirbelgelenke) zu erlangen. Allgemeine Nebenwirkungen werden damit begünstigt.So far, it has therefore been common to use systemic high To treat tissue levels in order to be effective  ve therapeutic dose in the target structure (volume disk u. Vertebral joints). General This favors side effects.

Das gleiche gilt auch für die Therapie von Nerven­ erkrankungen. Bedingt durch die Ausbildung der Bluthirnschranke sind Anwendungen großer therap. Proteine sehr erschwert.The same applies to the therapy of nerves diseases. Due to the training of Blood brain barrier are applications of great therap. Proteins very difficult.

2. Gentherapie bei Wirbelsäulenerkrankungen2. Gene therapy for spinal disorders

Das grundlegende therapeutische Konzept des mo­ lekularbiologischen Ansatzes des Patentes liegt darin, den Gencode der Chondrozyten, Fibrobla­ sten und Nervenzellen der Wirbelsäule so zu verän­ dern, daß diese Zellen Proteine mit therapeutischen Eigenschaften synthetisieren (Beispiel Wirbelgelen­ ke Abb. 1). Im Falle der Expression der Gene syn­ thetisieren und sezernieren Chondrozyten, Neuro­ ne und Fibroblasten antiinflammatorische und anal­ getische Moleküle in den Bandscheibenraum oder in den Gelenkinnenraum.The basic therapeutic concept of the molecular biological approach of the patent is to change the gene code of the chondrocytes, fibroblasts and nerve cells of the spine so that these cells synthesize proteins with therapeutic properties (example vertebral gels ke Fig. 1). If the genes are expressed, chondrocytes, neurons and fibroblasts synthesize and secrete anti-inflammatory and analgesic molecules into the intervertebral disc space or into the interior of the joint.

Durch diesen Zugang kann das Repertoire der z.Z. therapeutisch genutzten relativ kleinen Moleküle auf neue größere Proteine ausgeweitet werden. Dies wäre von Vorteil, da einige Proteine (z. B. Hemm­ stoffe der Zytokine) Eigenschaften aufweisen, die sie für die o.g. Erkrankungen favorisieren. Wegen ihrer Größe, schlechten Penetration in die bradytro­ phen Gewebe und wegen ihrer physiologischen In­ stabilität wurden bisher solche Substanzen auf ih­ ren praktischen therapeutischen Nutzen nur wenig untersucht.Through this access, the repertoire of the currently relatively small molecules used for therapeutic purposes new larger proteins are being expanded. This would be advantageous because some proteins (e.g. inhibitor substances of the cytokines) have properties that them for the above Favor diseases. Because of their size, poor penetration into the bradytro phen tissue and because of their physiological in So far such substances have been found on ih little practical therapeutic benefit examined.

Der jetzige wissenschaftliche Stand in der Diskus­ sion der biochemischen Verursachung degenerati­ ver Erkrankungen des Bewegungsapparates läßt den Schluß zu, daß Interleukin-1 (IL-1) der ent­ scheidende Mediator der pathologischen Verände­ rungen ist (3, 10). IL-1 ruft synoviale Entzündung, Knorpelverlust und Knochenresorption hervor (2, 5, 6, 7). In tierexperimentellen Untersuchungen konnte gezeigt werden, daß Antikörper gegen IL-1 den Ausprägungsgrad der experimentellen Arthritis we­ sentlich abschwächen (10). Auch erste klinische Untersuchungen zur Behandlung der Arthrose mit IL-1 Rezeptorantagonisten (IRAP) erscheinen viel­ versprechend (1, 4). Im Zusammenhang degenera­ tiver Wirbelsäulenerkrankungen ist das Zusammen­ spiel der Interleukine mit dem peripheren Nervensy­ stem von besonderem Interesse (9).The current state of science in the discus sion of biochemical causation degenerati ver diseases of the musculoskeletal system concluded that interleukin-1 (IL-1) der ent outgoing mediator of pathological changes is (3, 10). IL-1 causes synovial inflammation, Cartilage loss and bone resorption (2, 5, 6, 7). In animal experiments, are shown that antibodies against IL-1 den Degree of expression of experimental arthritis we weaken considerably (10). Also first clinical Studies on the treatment of arthrosis with  IL-1 receptor antagonists (IRAP) appear a lot promising (1, 4). In the context of degenera tive diseases of the spine is together Interleukins play with the peripheral nerve system stem of special interest (9).

Mit Blick auf diesen Hintergrund wird vom Erfinder vorgeschlagen, ein Gentransfersystem bei degene­ rativen Wirbelsäulenerkrankungen und degenerati­ ven Nervenerkrankungen einzuführen,das insbe­ sondere die Wirkungen von IL-1 mit einem neuarti­ gen Gentransfersystem anatagonisiert. Dabei konnte entweder das Gen für IRAP oder für einen IL-1 Rezeptor in Fibroblasten, Chondrozyten und Neuronen codiert werden. Mit diesem gentechni­ schen Transfersystem lassen sich aber auch zu­ sätzliche oder alternative Gene für andere thera­ peutische Proteine in die Wirbelgelenke, Nerven oder in den Bandscheibenraum einbringen.With this in mind, the inventor proposed a gene transfer system at degene ratative spinal diseases and degenerati to introduce venous nerve disorders, especially especially the effects of IL-1 with a novel anatagonized gene transfer system. Here could either be the gene for IRAP or for one IL-1 receptor in fibroblasts, chondrocytes and Neurons are encoded. With this gentechni However, transfer systems can also be used additional or alternative genes for other thera peutic proteins in the vertebral joints, nerves or insert into the intervertebral disc space.

3. Strategien des Gentransfers3. Strategies of gene transfer

2 verschiedene Systeme werden vom Erfinder vor­ geschlagen(Abb. 2).The inventor proposed 2 different systems ( Fig. 2).

Der direkte Zugang wird durch Injektion eines Vektors in die kleinen Wirbelgelenke, in die Band­ scheibe oder in den peripheren Nerven möglich, der Fibroblasten, Chondrozyten und Nervenzellen in situ überträgt (linke Seite Abb. 2).Direct access is possible by injecting a vector into the small vertebral joints, into the intervertebral disc or into the peripheral nerves, which transmit fibroblasts, chondrocytes and nerve cells in situ (left side Fig. 2).

Beim indirekten Zugang wird Bandscheibengewe­ be, Fibroblasten und Chondrozyten der kleinen Wir­ belgelenke oder Nerv entnommen, diese Zellen in vitro verändert und in das Entnahmegebiet retrans­ plantiert (rechte Seite Abb. 2).With indirect access, intervertebral disc tissue, fibroblasts and chondrocytes are removed from the small vertebral joints or nerve, these cells are changed in vitro and retransplanted into the removal area (right side Fig. 2).

Mit Hinblick auf die Entwicklung in der Endoskopie erscheint der indirekte Zugang für einen chirur­ gisch klinischen Einsatz besonders elegant, wäh­ rend der direkte Zugang technisch einfacher durch­ zuführen ist und eine allgemeine klinische Anwen­ dung damit erleichtert wird.With regard to the development in endoscopy indirect access for a surgeon appears clinically particularly elegant, wuh rend the direct access technically easier is a general clinical application  This will make it easier.

Der direkte Zugang wird aber durch die Unfähigkeit des retroviralen Vektors erschwert, ruhende Zellen in situ zu infizieren, da Retroviren Zellteilung zur In­ fektion benötigen. Allerdings kann die Zellteilung durch Entzündung, Verletzung oder partielle Gewe­ beentnahme selbst angeregt werden. Andere Vek­ toren (z. B. Adenoviren, Adeno-assoziierte Viren oder Herpesviren) infizieren auch nicht teilende Zel­ len, so daß diese Vektoren die vorgenannten Pro­ bleme umgehen könnten.However, direct access is due to inability of the retroviral vector complicates resting cells to be infected in situ, since retroviruses cell division to In need a fitting. However, cell division can due to inflammation, injury or partial tissue be stimulated yourself. Other Vek gates (e.g. adenoviruses, adeno-associated viruses or herpes viruses) also infect non-dividing cells len, so that these vectors the aforementioned Pro could handle bleme.

Beim jetzigen Stand der Entwicklung ist zu erwar­ ten, daß der indirekte Zugang als effektiver anzuse­ hen ist, da die Kotransduktion eines selektiven Mar­ kers eine Identifikation teilender Zellen ermöglicht.At the current stage of development is to be expected that indirect access should be viewed as more effective hen, since the co-transduction of a selective Mar kers enables identification of dividing cells.

4. Experimente4. Experiments

Nach Entnahme der Fibroblasten, Chondrozyten und Nerven aus den kleinen Wirbelgelenken, der Band­ scheibe und dem Nervenkanal im Tierexperiment wurden die angelegten Zellkulturen mit einem Re­ trovirus infiziert (BAG-Virus oder MFGLac-Z Virus) der Marker Gene für B-Galactosidase (lac Z) enthält und gegenüber dem Neomycin Analog (G 418 neo+) resistent ist.After removal of the fibroblasts, chondrocytes and Nerves from the small vertebral joints, the band disc and the nerve canal in animal experiments the created cell cultures with a Re trovirus infected (BAG virus or MFGLac-Z virus) the marker contains genes for B-galactosidase (lac Z) and compared to the neomycin analog (G 418 neo +) is resistant.

Diese neo-selektierten Zellen (Menge 106) wurden in den ursprünglichen Gewebebereich (Bandschei­ be, kleine Wirbelgelenke und Nerv) retransplan­ tiert, um die Persistenz und Expression dieser Ge­ ne in ihrer natürlichen Umgebung in vivo zu unter­ suchen.These neo-selected cells (amount 10 6 ) were retransplanted into the original tissue area (intervertebral disc, small vertebral joints and nerve) in order to investigate the persistence and expression of these genes in their natural environment in vivo.

12 Wochen nach Transplantation (s. Abb. 3 Histolo­ gie) konnte ein Überleben dieser gentechnisch ver­ änderten Zellen in ihrer natürlichen Umgebung be­ obachtet werden, erkennbar an der dunkelblauen Färbung der Zellen. Es zeigte sich außerdem eine regelrechte Kolonisation. Aus dem dann wieder ent­ nommenen Gewebe konnten diese veränderten Zellen in vitro erneut untersucht werden. Eine Ab­ stoßungsreaktion zeigte sich nicht. Die Experimente zeigen eindeutig, daß ein Marker-Gen in diese Zel­ len eingebracht werden kann, das sich in situ expri­ mieren kann.12 weeks after transplantation (see Fig. 3 histology), the survival of these genetically modified cells in their natural environment could be observed, recognizable by the dark blue color of the cells. There was also a real colonization. These modified cells could be examined again in vitro from the tissue then removed. There was no rejection reaction. The experiments clearly show that a marker gene can be introduced into these cells that can be expressed in situ.

Außerdem gelang es bei Fibroblasten, Chondrozyten und Neuronen den Gen-Code für einen Interleukin- 1 Rezeptorantagonisten in einen Retrovirus zu in­ korporieren. Die Zellen, die mit diesem Virus infi­ ziert wurden, waren in der Lage, Interleukin-1 Re­ zeptor Antagonisten zu produzieren.In addition, fibroblasts and chondrocytes were successful and neurons the gene code for an interleukin 1 receptor antagonists into a retrovirus corporation. The cells that infi with this virus were able to interleukin-1 Re to produce zeptor antagonists.

5. Klinische Perspektive5. Clinical perspective

Durch die gentechnisch mögliche Veränderung der Eigenschaften der Fibroblasten, Nervenzellen und Chondrozyten ergibt sich eine neuartige Strategie in der Behandlung schmerzhafter degenerativer Er­ krankungen der Bandscheibe (Abb. 5), des Nerven (Abb. 4) und der kleinen Wirbelgelenke (Abb. 5).The genetically engineered changes in the properties of the fibroblasts, nerve cells and chondrocytes result in a new strategy in the treatment of painful degenerative diseases of the intervertebral disc ( Fig. 5), the nerve ( Fig. 4) and the small vertebral joints ( Fig. 5).

Beim jetzigen Stand müssen die beschriebenen Verfahren verfeinert, ausgeweitet und auf ihre Si­ cherheit in größeren Serien untersucht werden.At the current status the described Process refined, expanded and based on your Si safety in larger series.

Besonders interessant erscheint therapeutisch die Möglichkeit, Gene mit antagonisierender Wirkung von IL-1 zu transduzieren.Therapeutically, this seems particularly interesting Possibility of genes with antagonizing effects to transduce from IL-1.

Die o.g. gentechnologischen Verfahren in Verbin­ dung mit Transplantationstechniken aus der Endo­ skopie (z. B. transarthroskopische Synovektomie und Diskektomie) werden einen innovativen Schub in der Behandlung dieser Erkrankungen erwarten lassen.The above genetic engineering processes in Verbin with transplantation techniques from Endo scopia (e.g. transarthroscopic synovectomy and discectomy) will be an innovative boost expect in the treatment of these diseases to let.

Da es sich bei dem Patentgegenstand prinzipiell um eine somatische Gentherapie handelt, ist ein Ver­ stoß gegen ethische Normen nicht gegeben. Since the subject matter of the patent is in principle somatic gene therapy is a ver does not violate ethical norms.  

Abb. 1 Das grundlegende therapeutische Konzept des molekularbiologischen Ansatzes liegt darin, den Gencode der Chondrozyten und der Fibroblasten der kleinen Wirbelgelenke und der Bandscheibe so zu verändern, daß diese Zellen Proteine mit therapeutischen Eigenschaften synthetisieren. Im Falle der Expression der Gene synthetisieren und sezernieren Chondrozyten und Fi­ broblasten (wie hier dargestellt) antiinflammatorische und analgetische Mole­ küle in den Gelenkinnenraum. Fig. 1 The basic therapeutic concept of the molecular biological approach is to change the gene code of the chondrocytes and the fibroblasts of the small vertebral joints and the intervertebral disc so that these cells synthesize proteins with therapeutic properties. If the genes are expressed, chondrocytes and fi broblasts (as shown here) synthesize and secrete anti-inflammatory and analgesic molecules into the interior of the joint.

Abb. 2 direkter und indirekter Zugang am Beispiel der kleinen Wirbelgelenke. Fig. 2 direct and indirect access using the example of the small vertebral joints.

Der direkte Zugang wird durch Injektion eines Vektors in die kleinen Wirbelgelenke, in die Bandscheibe oder in den peripheren Nerven möglich, der Fibroblasten, Chondrozyten und Nervenzellen in situ überträgt (linke Seite Abb. 2).Direct access is possible by injecting a vector into the small vertebral joints, into the intervertebral disc or into the peripheral nerves, which transmits fibroblasts, chondrocytes and nerve cells in situ (left side Fig. 2).

Beim indirekten Zugang wird Bandscheibengewebe, Fibroblasten und Chondrozyten der kleinen Wirbelgelenke oder Nerv entnommen, diese Zellen in vitro verändert und in das Entnahmegebiet retransplantiert (rechte Seite Abb. 2). With indirect access, intervertebral disc tissue, fibroblasts and chondrocytes are removed from the small vertebral joints or nerve, these cells are changed in vitro and retransplanted into the removal area (right side Fig. 2).

Abb. 3 (Histologie): Histologische Darstellung von Fibroblasten der Bandscheibe des Kaninchen nach Transplantation gentechnisch veränderter Zellen. Die dunkel gefärb­ ten Gewebeanteile entsprechen Fibroblasten, die zuvor außerhalb des Kniegelenkes gentechnisch verändert und retransplantiert wurden. Fig. 3 (histology): histological representation of fibroblasts of the rabbit intervertebral disc after transplantation of genetically modified cells. The dark-colored parts of the tissue correspond to fibroblasts that had previously been genetically modified and retransplanted outside the knee joint.

12 Wochen nach Transplantation (s. Abb. 3 Histologie) konnte ein Überleben dieser gentechnisch veränderten Zellen in ihrer natürlichen Umgebung beobachtet werden, erkennbar an der dunkelblauen Färbung der Zellen. Es zeigte sich außerdem eine re­ gelrechte Kolonisation. Aus dem dann wieder entnommenen Gewebe konnten diese veränderten Zellen in vitro erneut untersucht werden. Eine Abstoßungsreaktion zeigte sich nicht. Die Experimente zeigen eindeutig, daß ein Marker-Gen in diese Zellen ein­ gebracht werden kann, das sich in situ exprimieren kann. A survival of these genetically modified cells in their natural environment could be observed 12 weeks after the transplantation (see Fig. 3 histology), recognizable by the dark blue color of the cells. There was also regular colonization. These modified cells could be examined again in vitro from the tissue then removed. There was no rejection reaction. The experiments clearly show that a marker gene can be introduced into these cells that can be expressed in situ.

Abb. 4 Nerv: Anwendung der Erfindung bei Erkrankungen des Nerven. Therapeutische Gene werden in Makrophagen oder in Fibroblasten eingebracht, die dann therapeutische Proteine exprimieren. Fig. 4 Nerve: application of the invention to diseases of the nerve. Therapeutic genes are introduced into macrophages or into fibroblasts, which then express therapeutic proteins.

Abb. 5 Wirbelsäule: Bildliche Darstellung der Anwendung der Erfin­ dung, Möglichkeiten des Gentransfers an der Wirbelsäule (s. Text). Fig. 5 Spine: Pictorial representation of the application of the invention, possibilities of gene transfer to the spine (see text).

Literaturverzeichnis GentherapieBibliography of gene therapy

1. Carter DB et al. (1990)
Purification, cloning, expression and biological characterization of an interleukin-1 receptor antagonist protein. Nature 344, 633-638
2. Dingle JT, Saklatvala J, Hembry R, Tyler J, Fell HB, Jubb R (1979)
A cartilage catabolic factor from synovium. Biochem. J. 184, 177-180
3. Firestein GS, Alvaro-Garcis JM, Maki R (1990)
Quantitative analysis of cytokine gene expression in rheumatoid arthritis. J. Immunol. 144, 3347-3353
4. Hannum CH et al (1990)
Interleukin-1 receptor antagonist activity of a human interleukin-1 inhibitor. Nature 343, 336-340
5. Hubbard JR, Steinberg JJ, Bednar MS, Sledge CB (1988)
Effect of purified human interleukin-1 on cartilage degradation. J. Orthop. Res. 6, 180-187
6. Pettipher ER, Higgs GA, Henderson B (1986)
Interleukin-1 induces leukocyte infiltration and cartilage proteoglycan degradation in the synovial joint. Proc. Natl. Acad. Sci. USA 83, 8749-8753
7. Stashenko P, Dewhirst FE, Peros WJ, Kent RL, Ago JM (1987)
Synergistic interactions between interleukin-1, tumor necrosis factor, and lymphotoxin in bone resorption. J. Immunol. 138, 1464-1468
8. Van den Berg WB, Van de Loo FAJ, Otterness I, Arntz O, Jooster LAB (1991)
In vivo evidence for a key role of IL-1 in cartilage destruction in experimental arthritis. In Drugs in Inflammation. M. J. Parnham et al., eds. (Birkhauser Verlag, Basel, Schweiz) 159-164
9. Wehling P, Bandara G, Evans CH (1989)
Synovial cytokines impair the function of the sciatic nerve in rats: a possible element in the pathophysiology of radicular syndromes. Neuro Ortop. 7, 55-59
10. Wood DD, Ihrie EJ, Hamerman D (1985)
Release of Interleukin-1 from human synovial tissue in vitro. Arthritis Rheum. 28, 853-8621. Carter DB et al. (1990)
Purification, cloning, expression and biological characterization of an interleukin-1 receptor antagonist protein. Nature 344, 633-638
2.Dingle JT, Saklatvala J, Hembry R, Tyler J, Fell HB, Jubb R (1979)
A cartilage catabolic factor from synovium. Biochem. J. 184, 177-180
3. Firestein GS, Alvaro-Garcis JM, Maki R (1990)
Quantitative analysis of cytokine gene expression in rheumatoid arthritis. J. Immunol. 144, 3347-3353
4. Hannum CH et al (1990)
Interleukin-1 receptor antagonist activity of a human interleukin-1 inhibitor. Nature 343, 336-340
5. Hubbard JR, Steinberg JJ, Bednar MS, Sledge CB (1988)
Effect of purified human interleukin-1 on cartilage degradation. J. Orthop. Res. 6, 180-187
6. Pettipher ER, Higgs GA, Henderson B (1986)
Interleukin-1 induces leukocyte infiltration and cartilage proteoglycan degradation in the synovial joint. Proc. Natl. Acad. Sci. USA 83, 8749-8753
7.Stashenko P, Dewhirst FE, Peros WJ, Kent RL, Ago JM (1987)
Synergistic interactions between interleukin-1, tumor necrosis factor, and lymphotoxin in bone absorption. J. Immunol. 138, 1464-1468
8. Van den Berg WB, Van de Loo FAJ, Otterness I, Arntz O, Jooster LAB (1991)
In vivo evidence for a key role of IL-1 in cartilage destruction in experimental arthritis. In drugs in inflammation. MJ Parnham et al., Eds. (Birkhauser Verlag, Basel, Switzerland) 159-164
9. Wehling P, Bandara G, Evans CH (1989)
Synovial cytokines impair the function of the sciatic nerve in rats: a possible element in the pathophysiology of radicular syndromes. Neuro Ortop. 7, 55-59
10. Wood DD, Ihrie EJ, Hamerman D (1985)
Release of Interleukin-1 from human synovial tissue in vitro. Arthritis rheum. 28, 853-862

Claims (10)

1. Methode eines Gentransfersystems zur Einschleusung therapeutischer Gene mit­ tels Vektoren in Körperzellen und anschließender davon abhängiger Expression thera­ peutischer Proteine und Sekretion dieser therapeutischen Proteine in den extrazellulä­ ren Bereich durch die gentechnisch veränderten Körperzellen.1. Method of a gene transfer system for the introduction of therapeutic genes with tels vectors in body cells and subsequent expression thera dependent peutischen proteins and secretion of these therapeutic proteins in the extracellular area through the genetically modified body cells. 2. Methode nach Anspruch 1, dadurch gekennzeichnete daß die Körperzellen Nerven­ zellen, immunkompetente Zellen, mesenchymale oder ektodermale Zellen sind.2. Method according to claim 1, characterized in that the body cells have nerves cells, immunocompetent cells, mesenchymal or ectodermal cells. 3. Methode nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß die Körperzellen periphere Nervenzellen, Makrophagen, Lymphozyten, Fibroblasten, Chondrozyten sind.3. Method according to claim 1 or 2, characterized in that the body cells peripheral nerve cells, macrophages, lymphocytes, fibroblasts, chondrocytes are. 4. Methode nach Anspruch 1 bis 3, dadurch gekennzeichnet, daß die exprimierten the­ rapeutischen Proteine entzündungshemmende, schmerzstillende, regenerierende, im­ munsteigernde, hypotensive, antidegenerative und antiarthrotische Eigenschaften aufweisen.4. The method according to claim 1 to 3, characterized in that the expressed the therapeutic proteins anti-inflammatory, analgesic, regenerating, in Mun-increasing, hypotensive, anti-degenerative and anti-arthrotic properties exhibit. 5. Methode nach Anspruch 1 bis 4, dadurch gekennzeichnet, daß die exprimierten the­ rapeutischen Proteine zur Gruppe der Zytokine, seiner Inhibitoren, zur Gruppe der Opiate, zur Gruppe der Prostaglandine und deren Hemmstoffe gehören. 5. The method according to claim 1 to 4, characterized in that the expressed the therapeutic proteins to the group of cytokines, its inhibitors, to the group of Opiates belong to the group of prostaglandins and their inhibitors.   6. Methode nach Anspruch 1 bis 5, dadurch gekennzeichnet, daß die exprimierten the­ rapeutischen Proteine zur Gruppe der Interleukininhibitoren gehören.6. The method according to claim 1 to 5, characterized in that the expressed the therapeutic proteins belong to the group of interleukin inhibitors. 7. Methode nach Anspruch 1 bis 6, dadurch gekennzeichnet, daß die exprimierten the­ rapeutischen Proteine zur Gruppe der Interleukin-1 Inhibitoren gehören.7. The method according to claim 1 to 6, characterized in that the expressed the therapeutic proteins belong to the group of interleukin-1 inhibitors. 8. Methode nach Anspruch 1-7, dadurch gekennzeichnet, daß die Gene durch einen Vektor in vivo direkt in das Zellgebiet eingebracht werden, und das der Vektor zur Gruppe der Retroviren, Adenoviren, Adeno-assoziierte Viren, Herpesviren oder Lipo­ somen gehört.8. The method according to claim 1-7, characterized in that the genes by one Vector can be introduced directly into the cell area in vivo, and that the vector for Group of retroviruses, adenoviruses, adeno-associated viruses, herpes viruses or lipo somen heard. 9. Methode nach Anspruch 1-8, dadurch gekennzeichnet, daß die zu verändernden Zellen aus dem Körper transplantiert werden, die teilungsfähigen Körperzellen in vitro selektioniert werden, die Gene durch einen Vektor in vitro direkt in die Zellen einge­ bracht werden, und daß der Vektor aus der Gruppe der Retroviren, Adenoviren, Ade­ no-assoziierte Viren, Herpesviren oder Liposomen gehört , und daß diese gentech­ nisch veränderten Zellen in den Körper retransplantiert werden und therapeutische Proteine exprimieren und in den extrazellulären Bereich sezernieren.9. The method according to claim 1-8, characterized in that the to be changed Cells are transplanted from the body, the divisible body cells in vitro are selected, the genes are inserted directly into the cells by a vector in vitro be brought, and that the vector from the group of retroviruses, adenoviruses, ade belongs to no-associated viruses, herpes viruses or liposomes, and that these are genetically engineered African cells are retransplanted into the body and therapeutic Express proteins and secrete them into the extracellular area. 10. Methode nach Anspruch 1-9, dadurch gekennzeichnet, daß insbesondere degene­ rative Erkrankungen der Wirbelsäule und der Nerven Gegenstand der erfundenen Methode sind.10. The method according to claim 1-9, characterized in that in particular degenerate rative diseases of the spine and nerves the subject of the invented Are method.
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