ADENOVIRAL- BASED BIOLOGICAL DELIVERY AND EXPRESSION SYSTEM FOR USE IN THE TREATMENT OF OSTEOARTHRITIS

Abstract

The invention relates to an adenoviral-based biological delivery and expression system for use in the treatment or prevention of osteoarthritis in human or mammalian joints by long-term inducible gene expression of human or mammalian interleukin-I receptor antagonist (IL-1 Ra) in synovial cells, comprising a helper-dependent adenoviral vector containing a nucleic acid sequence encoding for human or mammalian interleukin-I receptor antagonist (IL-I Ra), left and right inverted terminal repeats (L ITR and R ITR), the adenoviral packaging signal and non-viral, non-coding stuffer nucleic acid sequences, wherein the expression of the human or mammalian interleukin-I receptor antagonist (IL-I Ra) gene within synovial cells is regulated by an inflammation-inducible promoter.

Claims

1. An adenoviral based biological delivery and expression system for use in the treatment or prevention of osteoathritis in human or mammalian joints by long-term inducible gene expression of human or mammalian interleukin-1 receptor antagonist (Il-1Ra) synovial cells, comprising a helper-dependent adenoviral vector containing a nucleic acid sequence encoding for human or mammalian interleukin-1 receptor antagonist (Il-1Ra), left and right inverted terminal repeats (L ITR and R ITR), the adenoviral packaging signal and non-viral, non-coding stuffer nucleic acid sequences, wherein the expression of the human or mammalian interleukin-1 receptor antagonist (Il-1Ra) gene within synovial cells is regulated by an inflammation-inducible promoter, which is located upstream of the reading frame of the nucleic acid sequence encoding for human or mammalian interleukin-1 receptor antagonist (Il-1Ra) and which is specifically activated by increased levels of immune stimulatory substances.

2. The adenoviral-based biological delivery and expression system according to claim 1, wherein the inflammation-inducible promoter is selected from the group consisting of NF-κB promoter, interleukin 6 (Il-6) promoter, interleukin-1 (Il-1) promoter, tumor necrosis factor (TNF) promoter, cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3) promoter, serum amyloid A3 (SAA3) promoter, macrophage inflammatory protein-1α (MIP-1α) promoter, or hybrid constructs of the above.

3. The adenoviral-based biological delivery and expression system according to claim 1 or claim 2, wherein the helper-dependent adenoviral vector comprises a nucleic acid sequence set forth in SEQ ID NO 2 or SEQ ID NO 3, or a biologically effective part thereof.

4. The adenoviral-based biological delivery and expression system according to any one of the preceding claims, wherein the mammalian interleukin-1 receptor antagonist (Il-1Ra) is selected from the group consisting of murine Il-1Ra, equine Il-1Ra, canine Il-1Ra, cat Il-1Ra, rabbit Il-1Ra, hamster Il-1Ra, bovine Il-1Ra, camel Il-1Ra or their homologs in other mammalian species,

5. The adenoviral-based biological delivery and expression system according to any one of the preceding claims, wherein the helper-dependent adenoviral vector further comprises a marker gene that allows monitoring of the vector genome in the synovial cells.

6. The adenoviral-based biological delivery and expression system according to any one of the preceding claims, wherein the helper-dependent vector comprises a nucleic acid sequence set forth in SEQ ID NO 1 or a conserved sequence thereof encoding for the same amino acids.

7. The adenoviral-based biological delivery and expression system according to any one of the preceding claims, wherein the helper-dependent vector has at least 50%, 60%, 80%, 90% sequence homology with the nucleic acid sequence set forth in SEQ ID NO 1.

8. A pharmaceutical composition, comprising a helper-dependent adenoviral vector containing a nucleic acid sequence encoding for human or mammalian interleukin-1 receptor antagonist (Il-1Ra), left and right inverted terminal repeats (L ITR and R ITR), an adenoviral packaging signal and non-viral, non-coding stuffer nucleic acid sequences, wherein the expression of the human or mammalian interleukin-1 receptor antagonist (Il-1Ra) gene within synovial cells is regulated by an inflammation-inducible promoter, which is located upstream of the reading frame of the nucleic acid sequence encoding for human or mammalian interleukin-1 receptor antagonist (Il-1Ra) and which is specifically activated by increased levels of immune stimulatory substances, for the treatment or prevention of osteoathritis.

9. The pharmaceutical composition according to claim 8, wherein the inflammation-inducible promoter is selected from the group consisting of NF-κB promoter, interleukin 6 (Il-6) promoter, interleukin-1 (Il-1) promoter, tumor necrosis factor (TNF) promoter, cyclooxygenase 2 (COX-2) promoter, complement factor 3 (C3) promoter, serum amyloid A3 (SAA3) promoter, macrophage inflammatory protein-1α (MIP-1α) promoter, or hybrid constructs of the above.

10. The pharmaceutical composition according to claim 8 or claim 9, wherein the helper-dependent adenoviral vector comprises a nucleic acid sequence set forth in SEQ ID NO 2 or SEQ ID NO 3, or a biologically effective part thereof.

11. The pharmaceutical composition according to any one of the preceding claims, wherein the mammalian interleukin-1 receptor antagonist (Il-1Ra) is selected from the group consisting of murine Il-1Ra, equine Il-1Ra, canine Il-1Ra, cat Il-1Ra, rabbit Il-1Ra, hamster Il-1Ra, bovine Il-1Ra, camel Il-1Ra or their homologs in other mammalian species.

12. The pharmaceutical composition according to any one of the preceding claims, wherein the helper-dependent adenoviral vector further comprises a marker gene that allows monitoring of the vector genome in the synovial cells.

13. The pharmaceutical composition according to any one of the preceding claims, wherein the helper-dependent vector comprises a nucleic acid sequence set forth in SEQ ID NO 1 or a conserved sequence thereof encoding for the same amino acids.

14. The pharmaceutical composition according to any one of the preceding claims, wherein the helper-dependent vector has at least 50%, 60%, 80%, 90% sequence homology with the nucleic acid sequence set forth in SEQ ID NO 1.

15. Use of an adinoviral-based biological delivery and expression system according to any one of claims 1 to 7 for expressing interleukin-1 receptor antagonist (Il-1Ra) in synovial cells ex vivo.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure Legends

[0041] FIG. 1

[0042] The Figure shows a basic gene map of the helper-dependent adenoviral vector of the invention. The vector backbone consists of the left and right inverted terminal repeats (ITR), adenoviral packaging signal (Ψ) and non-coding, non-viral stuffer sequences (remaining unmarked sequence between ITRs). The cDNA murine Il-1Ra is cloned between the viral left and right ITRs of the used adenoviral vector. The gene of Il1-Ra is controlled by inflammation-inducible NF-κB5-ELAM promoter.

[0043] FIG. 2

[0044] A. Helper-dependent and first generation adenoviral vectors mediate the same level of marker gene expression. Mice were injected intra-articularly with 10.sup.8 virus particles (VP) of a luciferase expressing helper-dependent (HDAd-luc) or a respective first generation (Ad-luc) adenoviral vector. Three days later mice were imaged using IVIS 200 series imaging system (Caliper Life Sciences, Hopkintom Mass.). Strong bioluminescence signals were detected in the joints injected with both HDAd-luc and Ad-luc adenoviral vector. Both knee joints of four mice per group were injected; representative pictures of two mice of each group are shown.

[0045] B. Helper-dependent adenoviral vector mediates long-term marker gene expression in joints. Luciferase expression of the mice described in A was followed by repeated bioluminescence imaging and quantified using Living Image 2.5 software (Caliper Life Sciences). Expression decreased and was undetectable by 30 days with the first generation adenoviral vector (Ad-luc). With the helper-dependent adenoviral vector (HDAd-luc) expression also declined but plateaued at 10 days and has been around this level for 380 days.

[0046] FIG. 3

[0047] Helper-dependent adenoviral vector infects synoviocytes efficiently. Mice were injected intra-articularly with 10.sup.8 VP of a LacZ expressing HDAd. One day later, mice were sacrificed and LacZ staining on sectioned joints was performed. Strong expression (dark blue staining) was seen in the synovium while no staining could be observed in chondrocytes. Right picture is a higher magnification photograph (40×) of the framed area in the left picture (5×).

[0048] FIG. 4

[0049] Cells infected with HDAd-Il-1Ra produce large amounts of Il-1Ra. Human embryonic kidney cells (HEK293) were infected with 100 VP/cell of HDAd-Il-1Ra, HDAd-GFP or mock. Two days later Il-1Ra ELISA was performed with cell culture supernatant. Concentrations of about 700 pg/ml were measured for HDAd-Il-1Ra infected cells, whereas no Il-1Ra was detectable in the supernatant of HDAd-GFP or mock infected cells. To induce an inflammatory reaction, lipopolysaccharides (LPS, 100 ug/ml) were added to half of the samples and Il-1Ra concentrations were again determined one day later (day 4). Levels in HDAd-Il-1Ra samples increased to about 1600 pg/ml whereas uninduced cells produced less Il-1Ra compared to the previous day. No Il-1Ra expression was detected in any of the control samples (HDAd-GFP and mock).

[0050] FIG. 5

[0051] HDAd-Il-1Ra prevents the development of OA. Mice were injected intra-articularly into the knee joints with 10.sup.8 VP of HDAd-Il-1Ra, HDAd-GFP or mock and OA was induced by cruciate ligament transduction two days later. Mice were sacrificed after 4 weeks and joints were histologically prepared, sectioned and stained with Safranin O. A blinded pathologist evaluated the level of OA according to OARSI (Osteoarthritis Research Society International) standards (assignment of scores on a scale of 1-6, 1: no signs of OA at all, 6: maximum OA). Mice treated with HDAd-Il-1Ra had significantly lower OA scores compared with mice treated with HDAd-GFP and mock. (* indicates significant difference: p<0.05 by one-way ANOVA; n=10 joints per group).

[0052] FIG. 6

[0053] HDAd-Il-1Ra efficiently treats OA in mice.

[0054] A. HDAd-Il-1Ra treated joints have significantly lower OA scores compared to controls. OA was induced in mouse knee joints by cruciate ligament transection and the disease was allowed to develop. Two weeks after transaction, mice were in intra-articularly with 10.sup.8 VP HDAC-Il-1Ra, HDAd-GFP or mock. Mice were sacrificed 6 weeks later and joints were histologically prepared, sectioned and stained with Safranin O. A blinded pathologist evaluated the level of OA according to OARSI (Osteoarthritis Research Society International) standard (assignment of scores on a scale of 1-6, 1: no signs of OA at all, 6: maximum OA). Mice treated with HDAd-Il-1Ra had significantly lower OA scores compared with mice treated with HDAd-GFP and mock. No significant difference was found between the HDAd-Il-1Ra group and age matched, untransected (no OA induction) mice. (* indicates significant difference: p<0.05 by one-way ANOVA: n.fwdarw.8 joints per group).

[0055] B. HDAd-Il-1Ra treated joints demonstrate significantly higher cartilage volume compared to controls. Whole knee joints of mice treated the same way as described above were fixed in electron microscopy fixative and embedded in paraffin. Samples were scanned using X-radia microXCT scanner (Xradia, Pleasanton, Calif., USA) and was visualized at 4 micron resolution. Computational 3D reconstruction of joints was performed and cartilage volume and surface area were quantified semi-automatically using TRI BON software (RATOC System Engineering, Tokyo, Japan). Significantly higher cartilage volume was measured in HDAd-Il-1Ra treated joints in comparison to controls. HDAd-Il-1Ra joints had similar volumes as untransected (healthy) joints. (* indicates significant difference: p<0.05, one-way ANOVA, n=6 joints/group).

[0056] C. HDAd-Il-1Ra treated ts demonstrate significantly larger cartilage surface area compared to controls. Cartilage surface area was measured as described above. HDAd-Il-1Ra treatment resulted in significantly higher cartilage surface area compared to controls. Surface area of HDAd-Il-1Ra treated joints was similar to that of untransected (healthy) controls. (* indicates significant difference: p<0.05, one-way ANOVA, n=6 joints/group).