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 osteoathritis in human or mammalian joints by long-term inducible gene expression of human or mammalian interleukin-1 receptor antagonist (II-1 Ra) in synovial cells, comprising a helper-dependent adenoviral vector containing a nucleic acid sequence encoding for human or mammalian interleukin-1 receptor antagonist (II-1 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-1 receptor antagonist (II-1 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 (II-1Ra) in synovial cells, comprising a helper-dependent adenoviral vector containing a nucleic acid sequence encoding for human or mammalian interleukin-1 receptor antagonist (II-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 (II-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 (II-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 (II-6) promoter, interleukin-1 (II-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 (II-1Ra) is selected from the group consisting of murine II-1Ra, equine II-1Ra, canine II-1Ra, cat II-1Ra, rabbit II-1Ra, hamster II-1Ra, bovine II-1Ra, camel II-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 (II-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 (II-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 (II-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 (II-6) promoter, interleukin-1 (II-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 (II-1Ra) is selected from the group consisting of murine II-1Ra, equine II-1Ra, canine II-1Ra, cat II-1Ra, rabbit II-1Ra, hamster II-1Ra, bovine II-1Ra, camel II-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 adenoviral-based biological delivery and expression system according to any one of claims 1 to 7 for expressing interleukin-1 receptor antagonist (II-1Ra) in synovial cells ex vivo.

Description

EXAMPLES

[0027] High levels of II-1Ra were measured in supernatants of synovial cells that were infected with a helper-dependent adenoviral vector (HDAd) of the invention. As shown below, the induction of inflammation with lipopolysaccharide (LPS) led to a dramatic increase of II-1Ra concentration as compared with uninduced samples. No II-1Ra was detected in non-infected samples (mock) or samples infected with a control vector (HDAd-GFP). The experiments demonstrate that cells infected with HDAd-mII-1Ra can produce high levels of II-1-RA. It further shows that II-1Ra is efficiently secreted from those cells, and that inflammatory conditions activate the NF-κ5-ELAM promoter leading to increased II-1Ra levels.

Production of the Helper-Dependent Adenoviral Vector of the Invention

[0028] FIG. 1 shows gene maps of the HDAd vectors of the invention. The full vector sequence is shown in SEQ ID NO 2 OR SEQ ID NO 3. The only difference between the two vectors is that GQ-201 carries the equine variant of II-1Ra whereas HDAd-mII-1Ra has the murine II-1Ra variant. Both vectors contain the inflammation inducible NF-κB5-ELAM promoter upstream of the II-1Ra cDNA according to SEQ ID NO 1 as well as inverted terminal repeats (ITR) and an adenoviral packaging signal. The vectors were cloned by standard digestion/ligation reactions according to the following strategy. The luciferase cDNA in pNifty-luc, a plasmid that contains the luciferase cDNA driven by a NF-κB5-ELAM promoter, was excised with NcoI and NheI and cDNAs for equine or murine II-1Ra were ligated into this position. The NF-κB5-ELAM promoter—murine II-1Ra or NF-κB5-ELAM promoter—equine II-1Ra cassettes were excised with NotI and PacI or EcoRI and PacI, blunted and inserted into pLPBL shuttle plasmid, which had been linearized with SalI and blunted. The NF-κB5-ELAM promoter—murine II-1Ra or NF-κB5-ELAM promoter—equine II-1Ra cassettes were then excised with AscI, which flanks both sides of the multiple cloning site, and ligated into AscI linearized pΔ28 plasmid (Toietta, G., Pastore, L., Cerullo, V., Finegold, M., Beaudet, A. L., and Lee, B. (2002). Generation of helper-dependent adenoviral vectors by homologous recombination. Mol Ther 5, 204-210.), which yielded the genomic plasmids pΔ28-mII-1Ra and pΔ28-eqII-1Ra. These plasmids were digested with PmeI in order to linearize the vector, liberate the inverted terminal repeats and excise bacterial resistance genes. Vectors were rescued and amplified as described before using the helper-virus AdNG163R-2 and 116 cell factories (Palmer, D., and Ng, P. (2003). Improved system for helper-dependent adenoviral vector production. Mol Ther 8, 846-852 ; Suzuki, M., Cela, R., Clarke, C., Bertin, T. K., Mouriñio, S., and Lee, B. (2010). Large-scale production of high-quality helper-dependent adenoviral vectors using adherent cells in cell factories. Hum Gene Ther 21, 120-126.)

HDAd Mediates Long-Term Marker Gene Expression in Joints

[0029] In order to determine long-term gene expression for up to one year in joints, mice were injected intra-articularly with a helper-dependent adenoviral vector of the invention (HDAd) and, for comparison, a first generation adenovirus (Ad) vector expressing firefly luciferase (luc) under the control of a CMV promoter. Luc expression was followed over time using in vivo bioluminescence imaging. Strong initial luc signals were detected three days after injection with both vectors (FIG. 2A). Expression decreased with both vectors thereafter and was undetectable after one month with the first generation vector Ad-luc (FIG. 2B). However, HDAd-luc luciferase expression stabilized at day 10 and has been at this level for 380 days.

HDAd Transduces Synovial Cells Following Intraarticular Injection

[0030] To evaluate HDAd transduction in mouse joints in detail, mice were injected intra-articularly with a LacZ expressing HDAd. Strong LacZ expression was seen in the synovium, however, no expression could be observed in chondrocytes (FIG. 3). The inventors also analyzed the liver of these animals to assess whether virus escapes from the joints or is spilled during the injection. Most importantly, no detectable vector concentrations over background could be measured by quantitative PCR (data not shown). Therefore, the vector specifically locates in the joints and remains there, which is of great benefit in the treatment or prevention of an osteoarthritic condition since it suggests minimal side effects.

HDAd-II-1Ra Infected Cells Secrete II-1Ra

[0031] An HDAd expressing II-1Ra under the control of the inflammation inducible NF-κB5-ELAM promoter was generated and its functionality was tested in vitro. High levels of II-1Ra were measured in the supernatant of HDAd-II-1Ra infected cells on day 3 (FIG. 4). Induction of inflammation with lipopolysaccharide (LPS) led to a dramatic increase of II-1Ra concentration compared with uninduced samples. No II-1Ra was detected in non-infected samples (mock) or samples infected with a control vector (HDAd-GFP).

HDAd-II-1Ra Prevents the Development of OA in Mice

[0032] To assess whether an HDAd expressing II-1Ra is able to prevent the development of OA, knee joints of mice were injected intra-articularly with HDAd-II-1Ra or a GFP expressing control vector (HDAd-GFP). Two days after injection, cruciate ligament transection was performed to induce OA development This osteoarthritis model was developed in Dr. Brendan Lee's research group and validated in several experiments (Ruan, Z., Dawson, B., Jiang M. M., Gannon, F., Heggeness, M., Lee, B. (2012). Quantitative volumetric imaging of murine osteoarthritic cartilage by phase contrast micro-computed tomography, submitted). The model involves transection of anterior and posterior cruciate ligaments of the knee joints, which leads to development of severe OA. Mice were sacrificed one month after OA induction and joints were prepared histologically and stained with Safranin O. The development of OA was scored by a blinded pathologist 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). HDAd-II-1Ra treated joints had significantly lower OA scores than HDAd-GFP treated or untreated joints, suggesting that HDAd-II1Ra prevented the development of OA (FIG. 5). The control vector HDAd-GFP did not seem to have any effect on the development of OA since the average OA score was comparable to the score of the untreated group.

HDAd-mII-Ra Treats OA in a Murine Model of the Disease

[0033] The efficacy of HDAd-mII-1Ra in the treatment of OA was evaluated in the murine disease model described above. The model was used to assess whether HDAd-II1-Ra can efficiently treat OA. Therefore, OA was induced by cruciate ligament transection (except in the untransected group) and OA was allowed to develop for two weeks. HDAd-II-1Ra, the control vector (HDAd-GFP) or vehicle was then injected and mice were sacrificed to analyze the joints another six weeks later. HDAd-GFP treated and uninjected mice developed OA to the same extent with an average score of approximately 4.5 (FIG. 6A). However, HDAd-II-1Ra treated mice had significantly lower OA scores compared with HDAd-GFP and mock treated. No significant difference was found between HDAd-II-1Ra and untransected (OA-free) mice suggesting efficient treatment of the disease or its prevention. The inventors further evaluated the joints in this experiment by micro computer tomography (μCT) analysis. This technique combines high resolution (down to 0.5 micron) x-ray CT scanning with phase contrast optics, which enables visualization of cartilage in small animal joints. Three-dimensional reconstruction of joints and computational tissue analysis tools can be used to quantify several cartilage parameters such as volume and surface area. HDAd-II-1Ra treated joints demonstrated significantly higher cartilage volume compared with HDAd-GFP and mock treated joints (FIG. 6B). No significant difference was seen between the HDAd-II-1Ra and untransected (OA-free) groups. Furthermore, cartilage surface area was significantly larger in HDAd-II-1Ra treated mice compared with HDAd-GFP and mock groups (FIG. 6C), while no significant difference was seen between HDAd-II-1Ra and untransected (OA-free) joints.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure Legends

[0034] FIG. 1

[0035] 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 of murine II-1Ra is cloned between the viral left and right ITRs of the used adenoviral vector. The gene of II1-Ra is controlled by inflammation-inducible NF-κB5-ELAM promoter.

[0036] FIG. 2

[0037] 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.

[0038] 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.

[0039] FIG. 3

[0040] 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×).

[0041] FIG. 4

[0042] Cells infected with HDAd-II-1Ra produce large amounts of II-1Ra. Human embryonic kidney cells (HEK293) were infected with 100 VP/cell of HDAd-II-1Ra, HDAd-GFP or mock. Two days later II-1Ra ELISA was performed with cell culture supernatant. Concentrations of about 700 pg/ml were measured for HDAd-II-1Ra infected cells, whereas no II-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 II-1Ra concentrations were again determined one day later (day 4). Levels in HDAd-II-1Ra samples increased to about 1600 pg/ml whereas uninduced cells produced less II-1Ra compared to the previous day. No II-1Ra expression was detected in any of the control samples (HDAd-GFP and mock).

[0043] FIG. 5

[0044] HDAd-II-1Ra prevents the development of OA. Mice were injected intra-articularly into the knee joints with 10.sup.8 VP of HDAd-II-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-II-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).

[0045] FIG. 6

[0046] HDAd-II-1Ra efficiently treats OA in mice.

[0047] A. HDAd-II-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 transection, mice were injected intra-articularly with 10.sup.8 VP of HDAd-II-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-II-1Ra had significantly lower OA scores compared with mice treated with HDAd-GFP and mock. No significant difference was found between the HDAd-II-1Ra group and age matched, untransected (no OA induction) mice. (*indicates significant difference: p<0.05 by one-way ANOVA; n=8 joints per group).

[0048] B. HDAd-II-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-II-1Ra treated joints in comparison to controls. HDAd-II-1Ra joints had similar volumes as untransected (healthy) joints. (*indicates significant difference: p<0.05, one-way ANOVA, n=6 joints/group).

[0049] C. HDAd-II-1Ra treated joints demonstrate significantly larger cartilage surface area compared to controls. Cartilage surface area was measured as described above. HDAd-II-1Ra treatment resulted in significantly higher cartilage surface area compared to controls. Surface area of HDAd-II-1Ra treated joints was similar to that of untransected (healthy) controls. (*indicates significant difference: p<0.05, one-way ANOVA, n=6 joints/group).