EP1027076A2 - Use of vectors such as adenoviruses and/or adeno associated viruses and/or retroviruses and/or herpes simplex viruses and/or liposomes and/or plasmids as a vehicle for genetic information enabling mammal cells to produce agents for the treatment of bone pathologies - Google Patents

Abstract

Disclosed is the use of vectors such as adenoviruses and/or adeno associated viruses and/or retroviruses and/or Herpes Simplex viruses and/or liposomes and/or plasmids as agents which stimulate mammal cells in order to produce therapeutic proteins that act as medicaments on a cellular level for the genetic treatment of bone pathologies.

Description

Use of vectors such as adenoviruses and / or adeno-associated viruses and / or retroviruses and / or herpes simplex viruses and / or liposomes and / or piasmids as vehicles for genetic information to enable mammalian cells to produce agents for the treatment of bone pathogens.

The invention relates to the use of adenoviruses and / or adeno-associated viruses and / or retroviruses and / or herpes simplex viruses and / or liposomes and / or piasmids as vectors, respective vehicles for genetic information to enable mammalian cells to be therapeutically enabled, to produce proteins, effective as medicinal products, for correcting bone pathologies at the cellular level.

Adenoviruses are simple DNA viruses made up of double-stranded DNA and proteins, some of which are human-like and cause infections of the eye and respiratory tract. Many species induce tumors in experimental animals that do not match the natural host or can transform cells in vitro. Adenoviral vectors are modified adenoviruses that have lost the ability to replicate in vivo and their typical pathogenicity.

The adeno-associated virus is also known as such and is used, for example, to label cells, as described in more detail in WO 95/14232.

For the purposes of the present invention, retroviruses are understood to mean class 6 RNA viruses which contain single-stranded ribonucleic acid (RNA) and the enzyme reverse transcriptase. With the help of this enzyme, the RNA that has entered the host cell is translated into DNA and then, together with additional repetitive sequences to be integrated into the host genome and replicated with it as a so-called provirus. Retroviruses, which are used as vectors in genetic engineering, are replication-deficient and apathogenic with regard to their typical pathogenicity.

The present invention has for its object to provide a genetic treatment method for bone pathologies detailed below, which includes a special agent to enable mammalian cells to produce therapeutic proteins. The transmission of the genetic information is made possible by special vectors.

The invention thus relates to the use of vectors such as adenoviruses and / or adeno-associated viruses and / or retroviruses and / or herpes simplex viruses and / or liposomes and / or piasmids as mammalian cell-stimulating agents for the production of therapeutic, drug-acting proteins on cellular Level, for the treatment of bone pathologies.

According to a special embodiment of the present invention, the adenoviruses are adenoviral vectors. These are of the currently more than 40 known human adenovirus types, such as Ad2 and Ad5, which, transformed into the vector, can be used according to the invention. These are modified to reduce your in vivo replication ability and pathogenicity by removing the essential E-1 sequence. Instead of the E1 sequence and the nonessential E3 sequence, the cDNA which codes for the therapeutic genes and marker genes is incorporated into adenoviral vectors. When the virus enters the cell, the cDNA is deposited episomally in the nucleus. The encoded substances are then produced and expressed via the common protein synthesis apparatus of the cells.

According to another particular embodiment of the present invention, the adeno-associated viruses are an adeno-associated viral vectors. Examples of this are the adeno-associated virus of the wild type, which is described in the Journal of Virology February 1983, Vol. 45, pp. 555-564, in particular FIGS. 1 to 4 and the sections Materials and Methods and Results, to which express reference is made.

Recombinant retroviruses that are currently used as vectors in human gene therapy experiments all derive from the wild-type Moloney Murine Leukemia (MoMuLV) retrovirus. The recombinant viruses are structurally identical to the wild type of the retrovirus, but carry a genetically modified genome that codes for the therapeutic genes or the marker genes. Recombinant retroviruses can no longer replicate themselves in vivo, but are still infectious and integrate the genome into the DNA of the target cells. The pathogenic viral genes have been completely removed.

According to a further preferred embodiment, the vectors are herpes simplex virus vectors. These are neurotropic human viruses that also attack mature neurons and, as a wild-type virus, can cause latent-type infections. For example, the herpes simplex virus -1 is used as viral vectors in a replication-deficient, apathogenic form. A particular advantage of the herpes simplex virus vector is the ability to integrate particularly large foreign cDNA fragments.

According to a particular embodiment of the present invention, the liposomes are liposomal vectors which contain the genetic information for growth factors or cytokines, or their inhibitors, and which can be transferred to mammalian cells. With regard to the liposomal vectors, reference is made to the publication by Gao and Huang in Biochem Biophys Res Commun 179: 280 to 285, 1991.

According to another particular embodiment of the present invention, the piasmids are those which contain the genetic information for growth factors or cytokines, or their inhibitors, and which can be transferred to mammalian cells. Reference is made to the publication by Smith, Shepherd, et al. Regarding plasmids used as vectors. in Gene Therapy 3: 190-200, 1996. In the method described according to the invention, therapeutic proteins are generated at the cellular level, which are growth hormones, cytokines or cytokine inhibitors. Examples include: Transforming growth factor-ß (TGF-ß) e.g. TGF-ß 1-5, Bone morphogenetic proteins (BMP) e.g. BMP-2-7, or osteogenic protein-1 (OP-1) e.g. OP-1 , Insulin like growth factor (IGF) e.g. IGF-1 and -2, fibroblast growth factor (FGF) e.g. FGF-1 and -2, platelet derived growth factor (PDGF), tumor necrosis factor (TNF) e.g. TNF- β or TNF-α, interleukin-6 inhibitors, macrophage-colony stimulating factor (M-CSF) inhibitors, granulcyte / macrophage-colony stimulating factor (GM-CSF) inhibitors, interleukins such as interleukin-4, interleukin-10 and Interleukin-13.

According to a particular embodiment of the present invention, the mammalian cells are animal or human cells, in particular bone cells, bone marrow cells, connective tissue cells and / or muscle cells.

According to a particular embodiment of the present invention, the vectors which lead to the production of the medicament by the transfected cells are applied parenterally.

According to a particular embodiment of the present invention, the vectors which lead to the production of the medicament by the transfected cells are applied intraosseously.

According to a special embodiment of the present invention, the vectors which lead to the production of the medicament by the transfected cells are applied locally to the site of the bone pathology.

According to a particular embodiment of the present invention, hereinafter referred to as the ex vivo method, mammalian cells are removed from the body for further treatment, then transduced in vitro using the aforementioned vectors, cultured in a suitable medium and autologously reimplanted, after which these mammalian cells are located at the site of the bone pathology produce effective therapeutic proteins as medicinal products. According to a particular embodiment of the present invention, the bone pathologies are osteoporosis, local or systemic bone mass loss, bone substance loss or bone structure disorders, bone fractures with bone substance loss, defect fractures, or pseudathroses, bone defect states after operations, Sudek's disease, bone substance loss in endoprosthesis disorders, osteolyseal looseness, osteolyseal looseness, osteolyseal looseness, osteolyseal looseness rheumatic type, for example in rheumatoid arthritis and / or osteonecrosis.

According to a particular embodiment of the present invention, the healing of transplants, in particular ligamentous, bone or tendinous transplants, can also be used as bone pathology, e.g. in knee and shoulder surgery.

According to a particular embodiment of the present invention, the genetic information (cDNA) of the therapeutically active proteins Bone Morphogenetic Proteins 2-7 (BMP 2-7), transforming growth factor-ß (TGF-ß), fibroplast growth factors ( FGFs), insulin-like growth factors (IGFs), platelet derived growth factors (PDGFs), and vascular endothelial growth factor (VEGF), or the cDNA of cytokines or cytokine inhibitors, e.g. Interleukin-I receptor antagonist protein (IL-1Ra) or tumor necrosis factor-α (TNF-α) inhibitors.

According to a particular embodiment of the present invention, the genetic information (cDNA) for the formation of cytokine inhibitors, e.g. B. Interleukin-1 receptor antagonist (IL-1Ra) and sTNFαR (soluble tumor necrosis factor α receptor) and Interleukin-6 inhibitors or absorption-inhibiting cytokines such as Interleukin-10 used.

The present invention is now explained in more detail by figures. Show it:

1 shows a plot of the production of the therapeutic protein IL-1 Ra per 50,000 cells after 48 h [in pg] for osteoblastic cells transduced with MFG (samples 1-6, values of 9111.5 pg on average) compared to non-transduced osteoblastic cells ( Control 1a - 3a, values around 0 pg) and retroviral LacZ transduced osteoblastic cells (control 1b - 3b, values around 0 pg). The measurement was carried out by ELISA analysis.

Fig. 2 is a plot of the spontaneous production of alkaline phosphatase (ALP) by human osteoblastic cells (HOB) (9.5 units per 1,000,000 cells), as well as the positive controls with murine Osteogenensis imperfecta stem cells (OIM) after stimulation with BMP-2 (30 units per 1,000,000 cells). The negative controls with immortalized synovial fibroblasts (HIG-82) do not produce alkaline phosphatase. There is also no ALP in the acellular nutrient medium (no cells).

3 shows a plot of the decrease in dry weight of the humeri, tibiae and fibulae in mg in white Balb / C mice by an ovariectomy (OVX) 12 days postoperatively, in comparison to simulated operated mice (sham). By intraosseous application of 10 ⁹ pfu Ad-IL-1Ra (OVX-IL-1Ra) the bone mass loss could be reduced by about 50% compared to the untreated control group (OVX-LacZ).

4 shows a plot of the expression of the marker enzyme luciferase [units / g] in the white Balb / C mouse in the bone tissue, muscle tissue, lymph node tissue, and also easily and transiently in the liver, after intraosseous / intramuscular transduction of the femora with 10 ⁹ pfu ad -Luc. Lungs and spleen are apparently not reached by the adenoviral vectors and show no enzyme expression. In bones and muscles, enzyme expression lasts for the whole Examination period of 21 days, expression in the liver can only be determined for a period of five days, in the draining inguinal nymph nodes for a period of 14 days. A small and time-limited transgene expression can thus be assumed outside the application area.

5 shows the systemic detection of transgenic IRAP (interleukin-1 receptor antagonist protein) over a course of 12 days in white Balb / C mice in order to test the quality of various application forms of Ad-IL-1Ra in systemic bone diseases. After intrafemoral application (♦) of 10 ⁹ pfu Ad-IL-IRa, positive systemic IRAP levels were found for the entire examination period of 12 days with maximum values of almost 100 pg / ml on the 3rd day. In contrast, intravenous administration of Ad-IL-1Ra (•) leads to lower values of up to 32 pg / ml, with a maximum expression duration of 3 days. In the negative control, which was carried out with 10 ⁹ pfu of the non-therapeutic marker gene Ad-LacZ (A), systemic evidence for transgenic IL-1Ra levels could not be provided.

6 shows the amount of deoxypyridinoline crosslinks [nM] excreted in the urine after ovariectomy (OVX) or simulated ovariectomy (sham) in white Balb / C mice. It becomes clear that more deoxypyridinoline crosslinks are excreted in the urine after ovariectomy, which are considered to be direct osteoclast activity parameters.

7 shows a 50-fold enlarged view of a paraffin section of the intrafemoral cavity of Balb / C mice after intraosseous transduction with 10 ⁹ pfu adenoviral vectors which code for LacZ (β-galactosidase). Morphologically, primarily resting osteoblasts (lining osteoblasts) but also bone marrow cells are transduced. These cells are marked with arrows. 8 shows a plot of the expression of the transgenic enzyme luciferase [units / μl] after injection of 10 ¹⁰ pfu Ad-Luc into the femoral defect in the white New Zealand rabbit. The distribution pattern of the enzyme activity shows that after local application of vectors in the bone defect, the expression of the transgenes apparently mainly takes place from local tissue structures. Bones, defect / scar tissue and muscles express the transgene with high expression values (up to 70,000 units / 100 μl in the muscle). There is no evidence of transgenic activity in the lungs, spleen and in contralateral, musculoskeletal tissue samples (not shown graphically). Only in the liver can transient, weak transgenic activity be demonstrated for 5 days. The gene expression was longest detectable in the bone for a total of 42 days, in the defect-filling tissue and in the muscles no gene expression was detectable after 15 or 26 days.

9 shows a 50-fold magnification of a decalcified paraffin section from the area of the femoral defect on the white New Zealand rabbit. In addition to connective tissue cells, adipocytes and possibly stem cells are also morphologically transduced. Here, too, these cells are marked by arrows.

10 shows a 20-fold magnification of a decalcified paraffin section from the area of the defect edge in the femoral defect model on the white New Zealand rabbit. Histomorphologically, in addition to resting osteoblasts, which are indicated by arrows, connective tissue cells of the adjacent scar are also transduced.

11 shows the healing process of the femoral defect in the white New Zealand rabbit as an example on the basis of three X-ray images, 5 weeks after intralesional transduction with 2 × 10 ¹⁰ pfu adenoviral vectors which code for BMP-2. An almost complete filling of the defect chamber with mineralized bones can be clearly seen in all three cases. FIG. 12 shows the healing process of the femoral defect in the white New Zealand rabbit as an example using three X-ray images, 5 weeks after intralesional transduction with 2 × 10 ¹⁰ pfu adenoviral vectors which code for the non-therapeutic marker gene luciferase. Clear osseous substance defects can be seen in the defect chamber. The comparison with the course of healing after application of therapeutic vectors (FIG. 11) testifies to a good effectiveness of the transgenic BMP-2.

13 shows a production of human IL-1 Ra osteoblast cells following an in vitro transduction with Ad-IRAP after a multiplicity of infection of 1000. The highest level of expression was found 3 weeks after the transduction. The gene expression was detectable in vitro for 72 days.

The present invention will now be explained in more detail by means of exemplary embodiments, percentages in each case relating to percentages by weight.

Application example 1:

In vitro evaluation of an osteoporosis therapy with retroviral vectors based on human osteoblastic cell populations.

Material and methodology

The basis of the cell cultures was human cancellous bone, which was obtained from informed patients with therapeutic radial or ulnar resection osteotomy. Cells with articular red femoral heads, often with necrotic changes, were difficult to cultivate and had only a short survival time in vitro.

Isolation of the cells

After removal of the cortical portions, the remaining cancellous bone was cut up with pieces of approximately 1 mm ³ and subsequently put into culture. Fragments with remaining cortical components were treated overnight with 0.1% collagenase (Seromed, Berlin) in 25 cm ² cell culture bottles (Nunc, Denmark). The collagenase solution was then removed and the cancellous fragments washed 3 times with PBS (Seromed, Berlin). The connective tissue cells released by the collagenase digestion were thereby removed quantitatively. The bone fragments were then cultured in nutrient medium (RPMI Medium 1640 (CibcoBRL, Grand Island, NY, USA)) with 10% fetal calf serum (FBS) and 1% penicillin / streptomycin solution (CibcoBRL, Grand Island, NY, USA)). The cells were cultivated in an incubator at 37 ° C. with 5% carbon dioxide fumigation. After about 16 to 20 days, the cells began to colonize the surface of the cancellous bone and the bottom of the culture bottles. The cells reached confluence after a further 3 weeks and, after trypsinization with 2 ml of trypsin (trypsin, 0.25% (CibcoBRL, Grand Island, NY, USA)), were distributed with the corresponding analyzes on cell culture flasks and cell culture dishes.

Production of the virus particles

The production cell lines for the retroviral particles are based on a derivative of the NIH3T3 cell line (CRIP). The cell line contains genetically integrated viral env and gag genes. These allow the production of retroviral viruses from the MFG-IRAP and BAG proviruses. MFG-IRAP and CRIP-BAG are described in the publication by G. Bandara et al. Proc. Natl. Acad. Be. USA 90: 10764 to 10768 (1993) under the title Intraarticular expression of bioiogically active interleukin-1-receptor-antagonist protein by ex vivo gene transfer. Both cell lines produce amphotropic retroviral particles, so they have a broad host specificity. MFG-IRAP carries the cDNA of the human interleukin-I receptor antagonist (hlL-1 Ra).

BAG carries the bacterial ß-galactosinase gene (LacZ) and a neomycin resistance gene (neoR), which enables selection of the transfected cell. The viral long terminal repeat (LTR) serves as a promoter for the HLL-1 Ra cDNA and LacZ. The CRIP cells are in (DMEM) medium (CibcoBRL, Grand Island, NY, USA), 10% FBS (CibcoBRL, Grand Island, NY, USA) and 1% HEPES (CibcoBRL, Grand Island, NY, USA) in Incubator cultivated at 37 ° C and 5% carbon dioxide fumigation.

The supernatant containing the virus was collected daily from confluent cultures. After filtration through a 0.45 μm fine syringe filter (Sartorius AG, Germany), the vector suspension was stored at -80 ° C until use. MFG and BAG are both derived from the Moloney Murine Leukemia Virus (MoMuLV, as described by P. Wehling et al. In Spine 21: 931-935 (1996) and S. Wells et al. In Gene Therapy 2: 512-520 (1995)). MFG has been used as a vector in several gene therapy trials, including a human Phase I trial in rheumatoid arthritis. Both retroviruses are no longer autonomously replication-competent, which means that an independent multiplication of the retrovirus in vivo is not possible.

Transduction

The osteoblastic cells were removed from the cell culture bottles by means of trypsin digestion and placed in 12 well plates cell culture dishes with 30,000 cells per dish. After approximately 80% confluence was reached, the nutrient medium was removed and the cells were washed twice with 3 ml of physiological saline. The transduction was carried out with 1 ml of viral supernatant at a virus concentration of 1 × 10 ⁶ particles of retrovirus per ml of virus supernatant and with a cationic polymer, that is to say a copolymer of N, N, N ', N'-tetramethyl-1, 6-hexanediamine and 1, 3-dibromopropane modulated by the Abbott Corporation under the designation Polybrene ^®, which is marketed, inter alia, from Aldrich as a promoter. During the first hour of transduction, the cell culture dishes were slowly centrifuged at room temperature and incubated at about 500 RPM and then overnight under cell culture conditions. After 48 hours, the nutrient medium was replaced. The supernatant of the MFG-IRAP transduced cells was collected and frozen at -80 ° C for later IL-1Ra quantification.

Detection of LacZ transfection

48 hours after transduction, the cells were fixed and LacZ expression was prepared using X-gal staining as follows. The cells were first washed with 1 ml PBS solution (Seromed, Germany), then fixed with solution 1 (1 ml 0.5% gluturaldehyde in 49 ml PBS (Roth GmbH, Germany)) for 10 min at room temperature. The cell culture dishes were then washed twice for 10 min each with solution 2 (1 ml of PBS solution in 1 mM magnesium chloride). Then solution 2 was replaced by the substrate solution (solution 3) consisting of 1500 μl 5 mM K ₃ Fe (CN) ₆ and 5 mM K4Fe (CN) ₆ , 30 μl 1 M MgCl ₂ , 750 μl 1 mg / ml 5- Bromine-4.chlor-3-indolyl-ß-D-galactpyranoside (X-gal) and 27 ml PBS were exchanged and left in the cell culture dishes overnight. The next morning, solution 3 replaced by PBS and the typical blue staining of the cells quantified by light microscopy.

Quantification of IL-1 _Ra expression

IL-1Ra was determined by a commercial ELISA kit (Biosource, USA) according to the manufacturer's information.

Determination of alkaline phosphatase

The determination of the activity of the alkaline phosphatase (ALP) was carried out in the cell cultures in the first passage. Immortalized synovial fibroblasts (HIG-82) served as negative controls; Immortalized bone marrow stem cells from Osteogenesis inperfecta mice (OIM), which had previously been stimulated with recombinant BMP-2, served as positive controls. All cell cultures were subjected to a three-time freeze-thaw cycle at -80 ° C to destroy the cell membranes. The burst cells were then stored at a temperature of -80 ° C until further analysis. The activity of the alkaline phosphatase was analyzed using a commercial analysis kit from Sigma Diagnostics (Dorset, United Kingdom) according to the manufacturer's instructions and determined photometrically at a wavelength of 405 nm after 1, 2 and 30 min on a UV-Max device from Molecular Devices, UNITED STATES.

Results

X-gal coloring

The transduction of retro-LacZ was carried out simultaneously in three cell culture dishes. In all cell culture dishes, the blue staining of cells was achieved by the X-gal staining. The proportion of transduced cells in the total cell number was 60% according to light microscopic quantification. No selection of transduced cells was carried out.

Gene expression

A transduction of the cells with retro-IRAP was carried out in 6 cell culture dishes. The supernatant from all 6 cells showed in the analysis by ELISA that the cell populations with the cDNA for hlL-1 Ra transduced and the transgene was expressed with an average of 9111.5 pg per 50,000 cells and 48 hours with a standard deviation of 522.4 pg. The ELISA analysis of the supernatant of the three culture dishes with non-transduced cell cultures, insofar as the analysis of the supernatant of the cells transduced with retroviral LacZ, gave negative values, as shown in FIG. 1.

ASSAY for alkaline phosphatase

Alkaline phosphatase (ALP) is one of the first markers that is already synthesized by immature osteoblasts and during the osteoblastic maturation process. The cell populations used in this experiment spontaneously expressed alkaline phosphatase with an average of 9.5 units per 1,000,000 units with a standard deviation of 0.7 units. The immortalized synovial fibroblasts (HIG-82) serving as negative controls did not express ALP, in contrast to the BMP-2 stimulated immortalized OIM cells, which served as positive controls. OIM and HIG-82 cells were chosen as control cell populations, since the potency of the OIM cells after stimulation with rhBMP-2 ALP is known. Because of their ability to synthesize ALP, the cells were classified as an osteoblastic cell population. The results of the ALP activity test are shown in FIG. 2.

Example of use 2:

Use of adenoviral vectors to develop a therapy for estrogen deficiency-related osteoporosis in the model of the Balb / C mouse

The basis of these experiments is the known knowledge that an estrogen deficiency via a systemic increase in the cytokines interleukin-1 and TNFa leads to an activation of osteoclasts, which cause a generalized loss of bone mass through an increased osseous absorption. The theory behind the attempts to evaluate osteoporosis therapy using the Balb / C mouse model is that, as is known, a generalized bone mass loss occurs after ovariectomy, which reaches its maximum after about two weeks. The aim is to use adenoviral vectors that code for IL-1 Ra to partially prevent bone loss. Such a genetic A procedure for the therapy of the loss of bone mass caused by estrogen deficiency has so far neither been described nor evaluated experimentally.

All experimental animals were 6 weeks old at the time of the experiments. A total of five different test series were carried out.

First, the excretion of deoxy-pyridinoline crosslinks in the urine after ovariectomy (OVX) and after simulated ovariectomy (sham) was measured after 2 days. The crosslinks were analyzed using a commercial kit from Metra Biosystems, Inc. (CA, USA) according to the manufacturer's instructions. This series of experiments was used to evaluate the ovariectomy effect on bone resorption and to test the suitability of this analysis for differentiating bone mass loss after treatment with therapeutic vectors.

Secondly, it was tested with adenoviral vectors which code for the marker gene LacZ (Ad-LacZ) whether a transduction of bone and bone marrow by adenoviral vectors is possible at all. The vectors were applied intrafemorally, using a transcutaneous, intercondylar approach, which made it possible to apply 100μl of a suspension with physiological saline and 10 ⁹ pfu Ad-LacZ intraosseously. Controls were carried out with instillation of physiological saline without vectors. The immunohistochemical staining method (LacZ staining is described several times in this application) enables the color of the successfully transduced cells to be shown by intensive blue staining of the cells expressing LacZ.

In a third series of experiments, the duration of gene expression and the intracorporeal distribution of the vectors after intraosseous vector application were investigated using the administration of adenoviral markers which code for the marker enzyme luciferase using the Balb / C mouse model. Three mice were killed on day 2, 14 and 21 after the vector application and different tissue types in the area of the injection, as well as various internal organs for analysis for expression of the transgene. This was measured photometrically with the Autolumat® LB953 from Berthold, Germany, as described in Application Example 3, carried out on the enzyme activity of the transgenic luciferase.

Fourthly, the intraosseous vector application commonly carried out in the experiments was compared with the parenteral vector application in order to investigate the duration and intensity of systemic transgene levels in both methods. The intraosseous application of the vectors was carried out as described above, while the parenteral injection was carried out in the generously applied retroorbital sinus in mice. The systemic IL-1Ra levels were determined in the serum of the mice after repeated blood withdrawals from the retroorbital sinus. The blood samples were taken before the vector application and on days 1, 2, 3, 5, 9 and 12 after the vectors were injected. The IL-1 Ra levels were analyzed using commercial ELISA kits according to the manufacturer's instructions.

In a fifth step, the effect of the adenovirally transferred, therapeutically effective IL-1Ra on ovariectomy-induced bone mass loss was examined. For this purpose, four groups with at least 8 mice each were formed, an ovariectomy being carried out in two groups and the mice receiving either the therapeutic vector Ad-IL-1 Ra or the non-therapeutic marker gene vector Ad-LacZ. The two remaining groups were treated in the same way, but instead of ovariectomy, only a simulated operation (sham) was performed. The analysis was carried out by measuring the total dry weight of the non-manipulated humeri, tibiae and fibulae in order to be able to evaluate the systemic effect of the vector application. The dry weight was measured in mg after darticulation and complete fleshing of the bones and treatment of the bones in a 90% acetone-alcohol bath.

Results

The analysis of the excretion of the deoxy-pyridinoline crosslinks in the urine confirmed, as demonstrated in Figure 6, that there was an increase in the rate after ovariectomy osteoblastic activity comes with increasing bone resorption. The ovariectomy model on the Balb / C mouse appears to be used to determine the loss of bone mass and is suitable for evaluating a genetic therapy for estrogen deficiency-related osteoporosis.

As demonstrated in FIG. 7, a number of different cell types are successfully transduced after the intraosseous application of adenoviral vectors (arrows). After immunohistochemical X-gal staining, which enables the cells that express the transgene to be displayed, predominantly osteoblasts are transduced, but also cells of the bone marrow. Since osteoblasts mature under the influence of suitable growth factors, an extended expression of transgenes can be expected due to the great stability and the immunologically privileged location of these cells.

The enzyme activity of the luciferase after intraosseous vector application of Ad-Luc is shown in FIG. 4. It demonstrates that transgene expression persists in the bones and muscles over the entire course of the examination interval of 21 days. Transgene expression from the liver (2 days) and draining lymph nodes (14 days) is transient and does not reach the level of enzyme activity in the area of the injection.

The comparison of the intrafemoral vector application, which is preferably used in these test series, with an intravenous vector application is presented in FIG. The figure shows that the intrafemoral application form is superior to the intravenous application form both in the intensity of the expression of transgenes and in the duration of the expression.

FIG. 3 demonstrates on the basis of the dry weights [mg] of the humeri, tibiae and fibulae that the ovariectomy-induced loss of bone mass, expressed here as the weight of mineralized bone, by genetic therapy with adenoviral vectors which code for IL-1 Ra by about 50 % can be reduced. Application example 3:

Accelerated bone growth by using adenoviral vectors, which code for growth factors, on the model of the white New Zealand rabbit.

Material and methodology

Animals:

Female New Zealand white rabbits over 6 months of age and weighing 4.3 to 5 kg were used in the study. In all animals, surgical defects in the femur were created in such a way that they did not heal after 9 weeks without treatment.

Surgical procedure

After induction of general anesthesia with ketamine hydrochloride (Ketaject ^® ) (40 mg / kg / M) and xylasin (Xyla-ject®) (3 mg / kg / M), both thighs of the rabbit were shaved, disinfected with isopropyl alcohol and prepared in a sterile manner. General anesthesia was initiated, supplemented with isoflurane 0.8 to 1.5% (AErrane®), with 50 to 100% oxygen and 50% nitrous oxide. Additional local anesthesia with 2% lidocaine was used while the periosteum was detached from the femora, as this procedure caused rabbit movements even under general anesthesia. There was a longitudinal preparation of the soft tissues. The entire diaphysis of the femora was dissected and the periosteal layer was completely removed. A 7-hole DCP plate (Synthes, Colorado, USA) was then placed over the lateral femora and fixed with self-tapping 2.7 mm screws. An exact defect of 1.3 cm was created using a ball milling machine. In order to protect the osteosynthesis from the enormous bending forces when the rabbits "kicked" postoperatively, 3 cerclage wires were used to achieve additional fixation over the plate at the distal and proximal ends of the femora. To completely remove bone fragments and bone marrow parts, was rinsed several times with physiological saline solution. A defect chamber was created by refixing the muscles around the femoral defect. All soft tissue layers were closed meticulously and the skin was sutured intracutaneously. No additional splinting or plastering was necessary. Vectors

Recombinant first generation adenoviruses (E1 \ E3 ') were used deliberately for the transduction of the cells, one of which carried the LacZ gene (Ad-LacZ) and the other the luciferase gene (Ad-Luc). In any case, gene expression was stimulated by a human early promoter. For each defect, 0.5 ml of a suspension of 1 x 10 ¹⁰ particels of adenoviral vectors, either in suspension with a physiological saline solution or with purified collagen gel (Vitrogen Collagen Corporation, Palo Alto, California, USA) was injected. The contralateral femoral defects received either 0.5 ml of a physiological saline solution or 0.5 ml of collagen gel without virus particles.

293 cells were propagated to produce the vectors Ad-LacZ and Ad-Luc. This method is described by Graham et al. in J. Gen Virol 36: 59-72 (1977). The 293 cells are each transfected with adenoviruses for 2 hours with a multiplicity of infection (moi) of 10. The cells are harvested after being scraped 36 to 48 hours after infection and resuspended in 5 ml of 50 mM Tris-Cl (pH 7.5) and 200 mM sodium chloride. After 5 freeze-thaw cycles, the viruses were purified using 2 cesium chloride step gradients (4 ° C., 30,000 rpm, 1 hour). Ad-LacZ was dialyzed against 10% glycerol dialysis buffer (100mM sodium chloride, 10mM Tris-Cl, pH 7.5; 1mM magnesium chloride, 10% v / v glycerin) as described by Mittereder et al. in J. Virol 70: 7498-7509 (1996) and Ad-Luc was dialyzed against 3% sucrose dialysis buffer (150 mM sodium chloride, 10 mM Tris-Cl, pH 7.5; 10 mM magnesium chloride, 3% v / v sucrose). Each of the vectors was titrated by its optical density at 260 nm, and both viruses were stored until use at a concentration of 7 x 10 ¹² particles per ml at -80 ° C.

Groups, data collection and analysis

Group 1: The femoral defect was created in 4 rabbits treated with 0.5% physiological saline containing 1 x 10 ¹⁰ particles of Ad-LacZ. Two rabbits were killed after two days, two more rabbits were killed 12 days after the operation. Group 2: Four rabbits were treated with a 0.5 ml mixture of collagen gel (Vitrogen) with 1 x 10 ¹⁰ particles of Ad-LacZ. The rabbits were killed on the schedule of Group 1.

Bone marrow, cortical and trabacular bone, scar tissue, which fills the defect and muscle, were analyzed for gene expression using the X-gal staining in all rabbits in groups 1 and 2. To determine whether there was systemic spread of the vectors, tissues were removed from the spleen, liver and lungs, as were bones, bone marrow and muscle from the contralateral untreated segmental defects. All tissue parts were fixed in 10% formaldehyde solution for 24 hours, then decalcified in 20% EDTA, within 2 to 3 weeks with a weekly exchange of the EDTA. After embedding the demineralized tissue in paraffin, blocks were cut and the tissue sections were stained with X-gal and eosin. The transduced cells were assessed histomorphologically by an independent pathologist.

Group 3: Eight rabbits from Group 3 were treated with 0.5 ml of a mixture of physiological saline with 1 x 10 ¹⁰ particles from Ad-Luc. The rabbits were put to sleep 2, 5, 10 and 14 days after the operation. Tissue samples were taken from the trabecular and cortical bone that surrounded the defect chamber, from the connective tissue that filled the defect chamber, and from the muscle that surrounded the femoral defect. In order to check a systemic distribution of the vectors, samples from the lungs, liver and spleen were analyzed as well as musculoskeletal samples from the non-transduced contralateral defect. All tissue samples were homogenized and, after adding 0.1 ml of physiological saline, subjected to a freeze-thaw cycle three times in order to destroy the cells. The cells were then stored at -80 ° C until the luciferase activity was measured. After the last thawing, the samples were centrifuged at 10,000 rpm (5600 xg) for 5 minutes and the supernatant solution was retained for the measurement of the luciferase activity. The luciferase activity was measured at room temperature using an AutoLumat® LB953 from Berthold, Germany in accordance with the manufacturer's instructions. Results

X-gal coloring

The histomorphological analysis showed that the production of Ad-LacZ, which was suspended either in physiological saline or in collagen gel, led to local transduction of bone, bone marrow, scar tissue and callus tissue as well as to transduction of the muscle surrounding the injection site (Fig . 9 to 10). In contrast, no LacZ ⁺ cells were found in the lungs, liver or spleen or in the contralateral femoral defect. When using collagen gel as a carrier for the adeno virus, the histological examination of the defect and the scar tissue, which filled the opening, showed a non-resorbed solid collagen structure even 12 days after the operation. From this one can only conclude that the adeno virus was encapsulated by the gel and therefore had no possibility to transduce. The histological analysis showed no evidence of local inflammation.

Luciferase assay

A different level of luciferase activity was found in the bone and bone marrow, as well as in the defect-filling connective tissue and in the surrounding muscle tissue at the injection site. 5 days after the transduction there was a slight luciferase activity in the liver which disappeared completely after 10 days. No luciferase activity could be found in the contralateral femur, the lungs and the spleen (Fig. 8).

Radiological course of healing after administration of therapeutic genes

After the preliminary tests with which the transducibility of the various musculoskeletal tissue structures had been demonstrated and the duration of the expression of transgenes had been determined, it had been shown that the femoral defect model on the New Zealand white rabbit is suitable for evaluating the effect of therapeutic genes on defect healing Using the same model, four and six rabbits were transduced with 1x10 ⁷ pfu Ad-TGF-ß or 2x10 ¹⁰ pfu Ad-BMP-2, simultaneously with four or five control rabbits of the same age, which transduced with the non-therapeutic marker gene Ad-Luc were. X-ray checks were carried out after the first check-up one week after surgery at two-weekly intervals. As shown in FIGS. 11 and 12, after 5 weeks in the group treated with Ad-BMP-2, a clear advantage in mineralization compared to the control group was visible. The final radiological examination in the 12th week showed a good osseous development of the femoral defect chamber in all 6 verum rabbits, in the case of poor development up to pseudarthrosis in the untreated control group. A similar trend was also seen in the group treated with Ad-TGF-ß. The radiological healing process showed significantly more mineralized substance in the femoral defect chamber compared to the control group, but without the formation of radiologically visible ossification in the sense of cortico-trabecular maturation. This result remained unchanged until the 16 postoperative week.

In conclusion, it can be concluded that Ad-BMP-2 had a stimulating effect on ossification in the femoral defect, Ad-TGF-b presumably more promotes non-specific mineralization of the tissue without leading to a final maturation of the bone. A combination of both vectors could possibly lead to a further acceleration of bone fracture healing in defect situations.

Biomechanics

Twelve weeks after the operation and injection of the viral vectors into the defect, the six rabbits treated with Ad-BMP-2 (2 × 10 ¹⁰ pfu) and 5 control rabbits, which received only 2 × 10 ¹⁰ pfu of the marker gene Ad-Luc, were treated had killed. The operated femora were largely fleshed out, sparing all ectopic bone bridges. Then the metal was carefully removed. It was shown that one femur of the control group was healed in connective tissue pseudoarthrosis, a second femur of the control group only showed such a thin bone bridge that it broke even with very careful preparation. This femur was therefore excluded from the evaluation. After removal of all metal, the preparations were frozen at -80 ° C. until further analysis.

The biomechanical analysis was carried out using the three-point bending method to determine the bending stiffness and the maximum bending force of the femora. The femora was stored frozen up to 24 hours before the tests and then slowly thawed in the refrigerator. Each femur was placed in the three-point bending system, which ensured a free bending distance of 4.5 cm. The load was taken in the posterior-anterior direction, with the load being transmitted at the center of the free bending path, which in each case represents approximately the center of the diaphyses.

The tests were performed with the Instron 8500 servo-hydralic testing system (Instron Corp., Canton, MA), an Instron 2500 Ibf load cell was used. The deformation of the femora was measured using the internal LVDT system. The data was collected using Instron's MAX software and analyzed using the MS-Excel software program.

A maximum deflection of 1 cm was used, with a bed rate of 0.5 cm / min. or 0.0833 mm / sec. The test was completed in the event of a fracture of the femora.

The verum group treated with Ad-BMP-2 differed significantly from the control group (p = 0.036) with regard to stiffness and also with regard to the force exerted (p = 0.0055).

Single measurements:

Stiffness force

Ad-BMP-2 control Ad-BMP-2 control

75.6000 80.8000 195.0000 102.0000

118.0000 64.0000 131.6000 103.2000

99.6000 53.2000 171.2000 82.0000

79.6000 0.0000 227.6000 0.0000

97.6000 206.4000

45.6000 92.4000

The biomechanical analysis according to the three-point bending method suggests that the use of the adenoviral vectors which code for BMP-2 and thus at the cellular level leads to the production of the active pharmaceutical ingredient Proteins cause an acceleration of bone defect healing can be achieved.

Histomorphometry

As in the experiments described above, the femoral defect model on the white New Zealand rabbit was used. 16 weeks after the operation of the animals and after injection 10 ⁷ pfu adenoviral vectors, the cDNA of which codes for the potentially therapeutic TGF-beta (Ad-TGF-beta), the four animals were sacrificed and prepared. Four white New Zealand rabbits, which received only 10 ⁷ pfu of a marker gene (Ad-Luc), served as a control group.

The image analysis was carried out as follows on the digitized histological section. The images were sharpened with a double band filter to define the blue-colored, bony areas (trichrome staining). Bony areas were defined using the color cube technique (3 x 3 pixels) after standardization against the normal cortical bone outside the defect had been carried out. The previous defect boundaries were digitally marked and the former area of interest (AOI) was analyzed for the intensity of the blue color. Four different measurements were made:

1. Absolute bone mass in the AOI

2. Medium light intensity - the measurement of the intensity of the staining enables conclusions to be drawn about the quality of the newly formed bone.

3. Mass of mineralized tissue in relation to the defect size in percent

4. IOD (integrated optical density), corresponds to the average optical light intensity per measured area

The following analyzed zones were determined as AOIs:

1. The entire area from the distal to the proximal screw hole

2. New bone formation along the removed DCP plate

3. New bone formation of the corticalis opposite the removed DCP plate

4. Sum of the defect edges

The total mass of mineralization in the defect area was more than twice that in the verum group treated with Ad-TGF-beta Control group (19.5 +/- 10.7 mm ² versus 9.04 +/- 4.3 mm ² ), but not significantly different. The differences were even clearer taking into account the area and the light intensity (IOP) (1754.0 +/- 906.0 mm ² versus 557.7 +/- 138.0 mm ² ). Here, too, the difference in the t-test was not significant for unconnected samples, which can be explained by the large scatter with small numbers of cases.

However, the results indicate that TGF-beta, produced and expressed at the cellular level, has a positive influence on mineralization and ossification after defect fractures. By using the technique of gene transfer with subsequent expression of the growth factor, which, as described in the preliminary experiments, is a predominantly local method, the known side effects of a systemic TGF-beta application could possibly be avoided.

Claims

claims

1. Use of vectors such as adenoviruses and / or adeno-associated viruses and / or retroviruses and or herpes simplex viruses and / or liposomes and / or piasmids as mammalian cell stimulating agents for the production of therapeutic proteins acting as drugs at the cellular level to the genetic level Treatment of bone pathologies.

2. Use according to claim 1, characterized in that the adenoviruses are adenoviral vectors.

3. Use according to claim 1, characterized in that the adeno-associated viruses are an adeno-associated viral vectors.

4. Use according to claim 1, characterized in that the retroviruses are retroviral vectors.

5. Use according to claim 1, characterized in that the liposomes are liposomal vectors which contain the genetic information for growth factors or cytokines, or their inhibitors.

6. Use according to claim 1, characterized in that the piasmids are those which contain the genetic information for growth factors or cytokines, or their inhibitors

7. Use according to any one of claims 1 to 6, characterized in that the therapeutic proteins are growth hormones, cytokines or cytokine inhibitors.

8. Use according to any one of claims 1 to 5, characterized in that the mammalian cells to be stimulated are animal or human cells, in particular bone cells, bone marrow cells, connective tissue cells and / or muscle cells.

9. Use according to any one of claims 1 to 8, characterized in that the vectors which lead to the production of the medicament by the transfected cells are administered parenterally.

10. Use according to any one of claims 1 to 8, characterized in that the vectors which lead to the production of the medicament by the transfected cells are applied intraosseously.

11. Use according to any one of claims 1 to 8, characterized in that the vectors which lead to the production of the medicament by the transfected cells are applied locally to the site of the bone pathology.

12. Use according to any one of claims 1 to 9, characterized in that for the treatment of mammalian cells removed from the body, then transfected in vitro by means of the aforementioned vectors, cultured in a suitable medium and re-implanted, then at the site of bone pathology, which as Medicinal products to produce therapeutic proteins after genetic manipulation of target cells.

13. Use according to any one of claims 1 to 12, characterized in that the bone pathologies are osteoporosis, local or systemic loss of bone mass, loss of bone substance or bone structure disorders, broken bones with loss of bone substance, defect fractures, or pseudarthrosis, bone defect states after operations, Sudek's disease, loss of bone substance Endoprosthesis loosening, peri-articular osteolysis in diseases of the rheumatic type and / or osteonecrosis.

14. Use according to any one of claims 1 to 12, characterized in that the bone pathology also includes the healing of grafts, in particular ligament, bone or tendinous grafts, e.g. in knee and shoulder surgery.

15. Use according to any one of claims 1-14, characterized in that to improve the bone structure, the genetic information (cDNA) of the therapeutic proteins BMP 2-7 and / or FGFs and / or IGFs and / or PDGFs and / or VEGF and / or TGF-ß is used or the cDNA of cytokines or cytokine inhibitors, e.g. hLL-1 Ra or TNF-α inhibitors.

16. Use according to any one of claims 1-14, characterized in that the genetic information (cDNA) for the formation of cytokine inhibitors, z. B. hLL-1Ra and sTNFαR and interleukin-6 inhibitors or absorption-inhibiting cytokines such as interieukin-10 can be used.

17. A method for treating bone pathologies using vectors such as adenoviruses and / or adeno-associated viruses and / or retroviruses and / or herpes simplex viruses and / or liposomes and / or piasmids as mammalian cell stimulating agents for the production of therapeutic proteins on cellular Level according to any one of the preceding claims 1 to 16.