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.

Description

FIELD OF THE INVENTION

[0001] The invention relates to the field of genetic engineering and provides an adenoviral-based biological delivery and expression system for use in the treatment of osteoarthritis in human or mammalian joints by long-term gene expression of human or mammalian interleukin-1 receptor antagonist (II-1Ra) in synovial cells.

DESCRIPTION OF THE BACKGROUND ART

[0002] Osteoarthritis (OA) is a degenerative joint disease that occurs in human or mammalian joints and constitutes a severe economical and medical problem (Matthews, G. L., and Hunter, D. J. (2011). Emerging drugs for osteoarthritis. Expert Opin. Emerging Drugs 1-13.; Brooks P M. Impact of osteoarthritis on individuals and society: how much disability? Social consequences and health economic implications. Curr Opin Rheumatol 2002; 14: 573-577). Cartilage is the tough connective tissue that covers the ends of bones in joints. It provides for a relatively frictionless, highly lubricated surface between rigid bones and allows for a smooth movement. During OA development, cartilage is partially or completely lost due to abnormal or excessive wearing, which leads to exposed bone ends that rub against each other resulting in inflammation, pain, swelling or loss of mobility. By now, the detailed reasons for the initial cartilage loss that leads to OA are not known, but there is a strong correlation between the incidence and age, obesity and joint overuse such as excessive athletic activity. Accordingly, OA is a major problem not only in humans, but in many mammals, in particular in horses that join in racings and show jumping.

[0003] Especially in horses, OA constitutes a significant problem with a tremendous economic impact, Horses spend almost their entire life on their legs, and those used for athletic purposes additionally undergo excessive training. Consequently, most of the joints in athletic horses are heavily overused which often results in lameness. Lameness accounts for about 70% of the cases where horses cannot participate in races or show jumping. About 60% of the cases can be directly linked to OA (Caron J P, Genovese, R L. Principals and practices of joint disease treatment. In: Ross M W And Dyson S, eds. Diagnosis and Management of Lameness in the Horse. 1.sup.st ed. Philadelphia: Saunders, 2003:746-764). Therefore, OA is the most common reason for the inability of a horse to participate in racing competitions or shows.

[0004] No curative treatment is currently available for OA--neither for horses, humans nor for any other mammalian species. Medical treatment is mostly aimed at alleviating the symptoms using analgesic drugs rather than establishing worn away cartilage. An analgesic treatment usually involves steroids and non-steroidal anti-inflammatory drugs (NSAIDS), which have shown efficacy in the treatment of OA for some decades. However, while these drugs can suppress joint inflammation, many of them are known to have deteriorating effects on the cartilage, which further worsens the underlying process of OA development. Hyaluronic acid, for instance, which restores viscoelasticity and lubrication of the joints, has also been widely used. Furthermore, polysulphated glycosaminoglycans injected into the joint or intramuscularly as well as orally administered glucosamine and chondroitin sulphate have shown some efficacy, however their mechanisms of action are not fully understood. Thus, currently used therapies have only limited efficacy in the treatment of OA and their success often depends on the severity of the case. Moreover, these drugs must be administered frequently, sometimes even in combination with each other. However, frequent drug injections into the joint are laborious, bear the risk for infections, cause stress for the horse and are costly. In addition, surgery has generally shown low efficacy in horses and is typically only performed in severe advanced-stage subjects. It follows that there is a clear and yet unmet medical need for more efficacious and sustained treatments that are at the same time also cost effective in the long run.

[0005] During OA, interleukin-1 (II-1) functions as a central mediator of inflammation (Dinarello C A. Interleukin-1 family. In: Thomson A W, Lotz M T (eds). The Cytokine Handbook. Academic Press: London, 2003, pp 643-668.;). Moreover, II-1 strongly inhibits matrix synthesis by cartilage and, at high concentrations, triggers matrix breakdown (Evans, C. H., Gouze, J. N., Gouze, E., Robbins, P. D., and Ghivizzani, S. C. (2004). Osteoarthritis gene therapy. Gene Ther 11, 379-389). To neutralize the effect of II-1 on synovial inflammation, treatment with interleukin-1 receptor antagonist (II-1Ra) constitutes a promising concept for treatment of affected osteoarthritic joints (Evans, C. H., Gouze, J. N., Gouze, E., Robbins, P. D., and Ghivizzani, S. C. (2004). Osteoarthritis gene therapy. Gene Ther 11, 379-389.; Caron J P et al. Chondroprotective effect of intraarticular injections of interleukin-1 receptor antagonist in experimental osteoarthritis. Suppression of collagenase-1 expression. Arthritis Rheum 1996; 39: 1535-1544). On nucleic acid level, II-1Ra is considerably conserved among mammalian species. For example, the cDNA sequences of human II-1Ra (Accession no: NM_173842) shares 82% homology with the murine variant (Accession no: NM_031167), 84% with the equine variant (Accession no: NM_001082525), 84% with the canine variant (Accession no: NM_001003096), 84% with the lapine variant (Accession no: NM_001082770) and 82% with the bovine variant (Accession no: NM_174357).

[0006] The basic concept of using gene therapy for the treatment of arthritis is well established (Evans C H, Robbins P D. Gene therapy for arthritis, In: Wolff J A (ed.). Gene Therapeutics: Methods and Applications of Direct Gene Transferm. Birkhauser: Boston, 1994, pp 320-343). In the closest prior art, the treatment of equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene using an adenoviral-mediated gene transfer has been described (D. D. Frisbie, S. C. Ghivizzani, P. D. Robbins, C. H. Evans, C. W. McIlwraith, Gene Ther 9, 12-20 (2002). The adenoviral vector used for expression of equine II-1Ra DNA was a first-generation adenoviral vector, which was shown to produce biologically active equine II-1Ra. Although clinical examinations of the horses in this study indicated that the therapeutic expression of II-1Ra significantly decreased signs of Joint pain as measured by the degree of lameness, the effect of delivery and expression of biologically active equine II-1Ra transgene was only short-term. Already 30 days following treatment of horses with equine II-1Ra by intra-articular injection of various amounts of the vector bearing II1-Ra, expression of equine II-1Ra in joints dropped to normal levels. Similar results were also detected in the US 2003/0091536 A1, which describes adenovirus particles encoding an interleukin-1 receptor antagonist for use in the treatment of joint disease. The adenovirus particles used were first generation adenoviral vectors.

[0007] A 2-component expression system consisting of C3-human immunodeficiency virus/transactivator of transcription [C3-Tat/HIV] with the constitutive cytomegalovirus (CMV) promoter in a polyarticular collagen-induced arthritis (CIA) model in mice has been described (BAKKER A C ET AL: "C3-Tat/HIV-regulated intraarticular human interleukin-1 receptor antagonist gene therapy results in efficient inhibition of collagen-induced arthritis superior to cytomegalovirus-regulated expression of the same transgene.", ARTHRITIS AND RHEUMATISM June 2002 LNKD-PUBMED:12115199, vol. 46, no. 6, June 2002 (2002-6), pages 1661-1670). This document specifically refers to rheumatoid arthritis (RA) as a chronic progressive autoimmune disease. It shows that collagen-induced arthritis (CIA) can be inhibited with high-systemic dosis of II-1Ra or with local production of II-1 Ra using an ex-vivo approach.

[0008] Helper-dependent adenoviruses (HDAd), also known as gutless or high-capacity adenoviruses, are the latest generation of adenoviral vectors (Mitani, K., Graham, F. L., Caskey, C. T. & Kochanek, S. Rescue, propagation, and partial purification of a helper virus-dependent adenovirus vector. Proc Natl Acad Sci USA 92, 3854-3858 (1995); Parks, R. J. et al. A Helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci USA 93, 13565-13570 (1996); Parks, R. J. Improvements in adenoviral vector technology: overcoming barriers for gene therapy. Clin. Genet. 58, 1-11 (2000)). These vectors are devoid of all viral sequences and are able to mediate long-term gene expression in various tissues (e.g. 7 years in the liver) in contrast to the more immunogenic first generation adenoviruses (Brunetti-Pierri, N. et al. Multi-Year Transgene Expression in Nonhuman Primates Following Hepatic Transduction with Helper-Dependent Adenoviral Vectors. American Society of Gene & Cell Therapy, Annual Meeting 2011 Molecular Therapy Volume 19, Supplement 1, May 2011). However, longevity of helper-dependent adenoviruses mediated gene expression in joints has not been evaluated to date.

[0009] Further helper-dependent adenoviral vector systems and their generation have also been described (PALMER DONNA ET AL: "Improved system for helper-dependent adenoviral vector production.",

MOLECULAR THERAPY: THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY November 2003 LNKD-PUBMED:14599819, vol. 8, no. 5, November 2003 (2003-11), pages 846-852, TOILEATTA GABRIELLE ET AL: "Generation of helper-dependent adenoviral vectors by homologous recombination.", MOLECULAR THERAPY: THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY February 2002 LNKD-PUBMED:11829528, vol. 5, no. 2, February 2002 (2002-02), pages 204-210).

[0010] The U.S. Pat. No. 5,747,072 A describes and claims a recombinant adenoviral vector having an expression control sequence operatively linked to a gene that encodes an anti-inflammatory polypeptide, ribozyme or antisense RNA molecule. Administering to the joint a therapeutically effect amount of a recombinant first generation adenoviral vector resulted in a reduced inflammatory response in the joint of the treated subject. Again, as in other studies, the long-term expression of II-1Ra was limited.

SUMMARY OF THE INVENTION

[0011] It is therefore an object of the present invention to provide an improved delivery and expression system that allows for a long-term expression of biologically active recombinant interleukin-1 receptor antagonist (II-1Ra) in synovial cells at human or mammalian joints for treatment and prevention of osteoarthritis. The solution for the problem is provided by an adenoviral-based biological delivery and expression system having the features as claimed in claim 1. Preferred embodiments of the invention are subject-matter of the dependent claims.

[0012] The adenoviral-based biological delivery and expression system according to the present invention is based on 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), adenoviral packaging signal and non-viral, non-coding stuffer nucleic acid sequences. Furthermore, the gene of the human or mammalian interleukin-1 receptor antagonist (II-1Ra) is controlled by an inflammation-inducible promoter. Preferred inflammation-regulated promoters for use in the present invention are NF-.kappa.B 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-1.alpha. (MIP-1.alpha.) promoter, or hybrid constructs of the above. The promoter sequences are upstream of the reading frame of the cloned II-1Ra gene. The inflammation-regulated promoter according to the present invention is specifically activated by increased levels of immune stimulatory substances such as lipopolysaccharide (LPS), which is a major component of the outer cell membrane of gram-negative bacteria. During osteoarthritis, a vartiety of immune stimulatory substances and cytokines are released, resulting in high levels of promoter-activating substances. One component, for instance, is NF-.kappa.B, which regulates the NF-.kappa.B promoter. Therefore, the release of such osteoarthritic-specific activators allows for the control of gene expression in joints of humans or mammals for treating or preventing an osteoarthritic condition.

[0013] The use of an inflammation-inducible promoter provides for specific control of II-1Ra gene expression in osteoarthritic tissue cells. Only cells that are affected by the disease will express and secrete the II-1Ra gene product, whereas cells that are not affected remain silent. The use of NF-.kappa.B5-ELAM promoter for inflammation-dependent gene expression is most preferred. However, any other inflammation-dependent promoter that result in a specific expression of the II-1Ra gene product in osteoarthritic tissue can be used in context of the present invention.

[0014] The helper-dependent adenoviral vector as used in the present invention minimizes immune responses in the host, and confers long-term gene expression of human or mammalian II-1Ra in joints that are affected by osteoarthritis.

[0015] To be most effective, the helper-dependent adenoviral vector of the invention is preferably administered in a single injection dose directly into the joints of the osteoarthritic subjects. Following intra-articular injection, the gene of II-1Ra is delivered to synovial cells such as synoviocytes. Synovial cells that are affected by inflammation start to produce recombinant II-1Ra protein under the control of the inflammation-inducible promoter such as the NF-.kappa.B promoter. High amounts of II-1Ra are then secreted into the joint space, where II-1Ra is able to inhibit inflammation and stop cartilage degradation by blocking the interleukin-1 receptor on the surface of synoviocytes and the cells embedded in the cartilage. Most importantly, high local concentrations of recombinant II-1Ra do not show any side effects.

[0016] Therefore, as shown in the examples below, pain, inflammation and cartilage degradation are inhibited effectively using the adenoviral-based biological delivery and expression system according to the present invention. High local and low systemic concentrations of the therapeutic protein II-1Ra are achieved, resulting in maximum efficacy in the treatment of OA at no or minimal side effects. It is further exemplified that cells containing the helper-dependent adenoviral vector of the invention are capable to produce recombinant II-1Ra for an extended period of at least one year. Consequently, medical and economic burden associated with frequent joint injections that were required in the known short-term treatments will be significantly reduced. In particular, the serious long-term side effects of commonly used steroids can be avoided by using the helper-dependent adenoviral vector of the invention due to its cartilage protective effect. Thus, common complications associated with OA treatment are minimized and joint health will be preserved in the long run resulting in sustained health improvement of the treated animal or human.

[0017] In addition, the inflammation-dependent II-1Ra production of the vector of the invention allows for the prevention of the development of an osteoarthritic condition as synovial cells that are infected with the adenoviral vector of the invention remain silent in the absence of immune stimulatory substances that could activate the NF-.kappa.B5-ELAM promoter or any other inflammation-dependent promoter. Only if the osteoarthritic condition initiates, the promoter is activated as a result of inflammation and subsequently II-1Ra is produced and secreted. Thus, by using the adenoviral delivery and expression system of the invention, this mechanism allows for the prevention of the development of osteoarthritis in an early stage.

[0018] An inflammation-dependent II-1Ra production of the vector of the invention can also be viewed as a safety feature to ensure that II-1Ra is no longer produced, for example when the osteoarthritic condition is resolved or has disappeared.

[0019] The helper-dependent adenoviral vector as used in the present invention does not carry any viral sequences, except the left and right inverted terminal repeats (ITRs) and the adenoviral packaging signal. Preferred helper-dependent adenoviral vectors to be used in the present invention are those based on the helper virus and helper-dependent backbone system developed by Palmer and Ng (Palmer, D., and Ng, P. (2003). Improved system for helper-dependent adenoviral vector production. Mol Ther 8, 846-852.) and Toietta et al (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.). A preferred adenoviral delivery and expression system according to the present invention comprises a nucleic acid sequens set forth in SEQ ID NO 2 or SEQ ID NO3, or a biologically effective part thereof. The nucleic acid sequence of SEQ ID NO 2 describes a murine helper-dependent adenoviral vector, and the sequence set forth in SEQ ID NO 3 describes a equine helper-dependent adenoviral vector, both bearing the murine and equine II-1Ra gene, respectively. Preferably, the system of the invention has at least 50%, 60%, 70%, 80%, 90% sequence homology with the vector set forth in SEQ ID NO 2 or SEQ ID NO 3.

[0020] "Biologically effective" in the context of the present invention means that the gene product of the adenoviral delivery and expression system comprises the full or partial polypeptide sequence of II-1Ra having the in-joint activity to neutralize the effect of II-1 on synovial inflammation.

[0021] The helper-dependent adenoviral vector of the invention preferably contains the cDNA sequence of II-1Ra that is controlled by the inflammation-inducible promoter. Although II-1Ra contains species-specific nucleic acid sequences, the adenoviral vector is able to express interleukin-1 receptor antagonist (II-1Ra) from any mammalian species or human. Preferably, the cDNA of the mammalian interleukin-1 receptor antagonist (II-1Ra) used for cloning is a cDNA 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.

[0022] In order to monitor the presence of genomic vector sequences in synovial cells, the helper-dependent adenoviral vector according to the invention preferably further comprises a marker gene that is visually or instrumentally detectable. Preferred marker genes are, for instance, green fluorescence protein (GFP) or luciferase enzyme.

[0023] As an example, the nucleic acid sequence of murine II-1Ra as used in the present invention is shown in the sequence listing set forth in SEQ ID NO 1. As noted above, any nucleic acid sequence resulting in a biologically active II-1Ra protein of any mammalian or human species can be used in the context of the present invention. Furthermore, also conserved nucleic acid sequences encoding for the same amino acids, polypeptide or protein fall under scope of the present invention. As illustrated in the prior art, II-1Ra genes of various species share a high homology among each other. Preferably, the helper-dependent adenoviral vector according to the invention contains a nucleic acid sequence (e.g. cDNA) of II-1Ra having at least 50%, 60%, 80%, 90% sequence homology with the nucleic acid sequence shown in SEQ ID NO 1. The invention also comprises biologically active nucleic acid sequences of II1-Ra or fragments thereof beside the full-length nucleic acid sequence of II1Ra.

[0024] The present invention further comprises 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), 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, for the treatment or prevention of osteoathritis. Preferred promoters as used in the context of the present invention are NF-.kappa.B 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-1.alpha. (MIP-1.alpha.) promoter, or hybrid constructs of the above.

[0025] It is a great benefit of the present invention that the adenoviral delivery and expression system specifically locates in the joints when administered intra-articularly. Most importantly, no measurable concentration of vector sequences could be detected in the liver of mice treated with the adenoviral system of the invention. Therefore, II-1Ra concentrations are expected to be highest in the joints injected with the vector of the invention while no significant side effects are expected in any other organ.

[0026] The invention will be further illustrated in the examples following below.

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-.kappa.5-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-.kappa.B5-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-.kappa.B5-ELAM promoter, was excised with NcoI and NheI and cDNAs for equine or murine II-1Ra were ligated into this position. The NF-.kappa.B5-ELAM promoter--murine II-1Ra or NF-.kappa.B5-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-.kappa.B5-ELAM promoter--murine II-1Ra or NF-.kappa.B5-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 p.DELTA.28 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 p.DELTA.28-mII-1Ra and p.DELTA.28-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., Mourinio, 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-.kappa.B5-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 (.mu.CT) 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 (.PSI.) 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-.kappa.B5-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.times.) of the framed area in the left picture (5.times.).

[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).

Sequence CWU 1

1

31535DNAArtificial Sequencemurine Il-1Ra 1atggaaatct gctggggacc ctacagtcac ctaatctctc tccttctcat ccttctgttt 60cattcagagg cagcctgccg cccttctggg aaaagaccct gcaagatgca agccttcaga 120atctgggata ctaaccagaa gaccttttac ctgagaaaca accagctcat tgctgggtac 180ttacaaggac caaatatcaa actagaagaa aagatagaca tggtgcctat tgaccttcat 240agtgtgttct tgggcatcca cgggggcaag ctgtgcctgt cttgtgccaa gtctggagat 300gatatcaagc tccagctgga ggaagttaac atcactgatc tgagcaagaa caaagaagaa 360gacaagcgct ttaccttcat ccgctctgag aaaggcccca ccaccagctt tgagtcagct 420gcctgtccag gatggttcct ctgcacaaca ctagaggctg accgtcctgt gagcctcacc 480aacacaccgg aagagcccct tatagtcacg aagttctact tccaggaaga ccaat 535229612DNAArtificial Sequencehelper-dependent adenoviral vector-murine Il-1Ra gene 2aaacatcatc aataatatac cttattttgg attgaagcca atatgataat gagggggtgg 60agtttgtgac gtggcgcggg gcgtgggaac ggggcgggtg acgtagtagt gtggcggaag 120tgtgatgttg caagtgtggc ggaacacatg taagcgacgg atgtggcaaa agtgacgttt 180ttggtgtgcg ccggtgtaca caggaagtga caattttcgc gcggttttag gcggatgttg 240tagtaaattt gggcgtaacc gagtaagatt tggccatttt cgcgggaaaa ctgaataaga 300ggaagtgaaa tctgaataat tttgtgttac tcatagcgcg taatatttgt ctagggccgc 360ggggactttg accgtttacg tggagactcg cccaggtgtt tttctcaggt gttttccgcg 420ttccgggtca aagttggcgt tttgatatca agcttatcga taccgtaaac aagtctttaa 480ttcaagcaag actttaacaa gttaaaagga gcttatgggt aggaagtagt gttatgatgt 540atgggcataa agggttttaa tgggatagtg aaaatgtcta taataatact taaatggctg 600cccaatcacc tacaggattg atgtaaacat ggaaaaggtc aaaaacttgg gtcactaaaa 660tagatgatta atggagagga tgaggttgat agttaaatgt agataagtgg tcttattctc 720aataaaaatg tgaacataag gcgagtttct acaaagatgg acaggactca ttcatgaaac 780agcaaaaact ggacatttgt tctaatcttt gaagagtatg aaaaattcct attttaaagg 840taaaacagta actcacagga aataccaacc caacataaaa tcagaaacaa tagtctaaag 900taataaaaat caaacgtttg cacgatcaaa ttatgaatga aattcactac taaaattcac 960actgattttg tttcatccac agtgtcaatg ttgtgatgca tttcaattgt gtgacacagg 1020cagactgtgg atcaaaagtg gtttctggtg cgacttactc tcttgagtat acctgcagtc 1080ccctttctta agtgtgttaa aaaaaaaggg ggatttcttc aattcgccaa tactctagct 1140ctccatgtgc tttctaggaa acaagtgtta acccacctta tttgtcaaac ctagctccaa 1200aggacttttg actccccaca aaccgatgta gctcaagaga gggtatctgt caccagtatg 1260tatagtgaaa aaagtatccc aagtcccaac agcaattcct aaaaggagtt tatttaaaaa 1320accacacaca cctgtaaaat aagtatatat cctccaaggt gactagtttt aaaaaaacag 1380tattggcttt gatgtaaagt actagtgaat atgttagaaa aatctcactg taaccaagtg 1440aaatgaaagc aagtatggtt tgcagagatt caaagaaaat ataagaaaac ctactgttgc 1500cactaaaaag aatcatatat taaatatact cacacaatag ctcttcagtc tgataaaatc 1560tacagtcata ggaatggatc tatcactatt tctattcagt gctttgatgt aatccagcag 1620gtcagcaaag aatttatagc cccccttgag cacacagagg gctacaatgt gatggcctcc 1680catctccttc atcacatctc gagcaagacg ttcagtccta cagaaataaa atcaggaatt 1740taatagaaag tttcatacat taaactttat aacaaacacc tcttagtcat taaacttcca 1800caccaacctg ggcaatatag tgagacccca tgcctgcaaa aaaaaaaaaa ttagccaggc 1860atggtagcat gtacctgtag tcccagctac ttgagaggtg aggtgggaaa atcactttag 1920tgcaggatgt tgaggctgga gtgaactgtg attgtgccac tgcactccag cctggacaat 1980agagcaagac cttgtctcaa aaaaatgcat taaaaatttt ttttaaatct tccacgtatc 2040acatcctttg ccctcatgtt tcataaggta aaaaatttga taccttcaaa aaaaccaagc 2100ataccactat cataattttt tttaaatgca aataaaaaca agataccatt ttcacctatc 2160agactggcag gttctgatta aatgaaattt tctggataat atacaatatt aagagagact 2220gtagaaactg ggccagtggc tcatgcctgt aatcccagca ctttgggagg ctgggtaaca 2280tggcgaaccc tgtttctaca aaataaaaat attagctggg agtggtggcg cacacctata 2340gtcccagcta ctcaggaggc tgaggtggaa ggatcgcttg aacccaggag gttgagactg 2400cagtgaactg tgatcattct gctgcactgc accccagcct gggcaacaga gaccttgtct 2460caaaaaaaaa aaaaaaagag acaaattgtg aagagaaagg tactctcata taacatcagg 2520agtataaaat gattcaactt cttagaggaa aatttggcaa taccaaaata ttcaataaac 2580tctttcccct tgacccagaa attccacttg aataaagctg aacaagtacc aaacatgtaa 2640aagaatgttt cttctagtac agtcggtaag aacaaaatag tgtctatcaa tagtggactg 2700gttaaatcag ttatggtatc tccataagac agaatgctat gcaaccttta aaatatatta 2760gatagctcta gacacactaa tattaaaagt gtccaataac atttaaaact atactcatac 2820gttaaaatat aaatgtatat atgtactttt gcatatagta tacatgcata ggccagtgct 2880tgagaagaaa tgtgtacaga aggctgaaag gagagaactt tagtcttctt gtttatggcc 2940tccatagtta gaatatttta taacacaaat attttgatat tataatttta aaataaaaac 3000acagaatagc cagacataca atgcaagcat tcaataccag gtaaggtttt tcactgtaat 3060tgacttaaca gaaaattttc aagctagatg tgcataataa taaaaatctg accttgcctt 3120catgtgattc agccccagtc cattaccctg tttaggactg agaaatgcaa gactctggct 3180agagttcctt cttccatctc ccttcaatgt ttactttgtt ctggtcccta cagagtccca 3240ctataccaca actgatacta agtaattagt aaggccctcc tcttttattt ttaataaaga 3300agattttaga aagcatcagt tatttaataa gttggcctag tttatgttca aatagcaagt 3360actcagaaca gctgctgatg tttgaaatta acacaagaaa aagtaaaaaa cctcatttta 3420agatcttact tacctgtcca taattagtcc atgaggaata aacacccttt ccaaatcctc 3480agcataatga ttaggtatgc aaaataaatc aaggtcataa cctggttcat catcactaat 3540ctgaaaaaga aatatagctg tttcaatgag agcattacag gatacaaaca tttgattgga 3600ttaagatgtt aaaaaataac cttagtctat cagagaaatt taggtgtaag atgatattag 3660taactgttaa ctttgtaggt atgataatga attatgtaag aaaacaacag gccgggcggg 3720ttggttcaca cgtgtaatcc cagcactttg ggaggctgag gcaggcagac tgcctgagct 3780caggagttcg agaccagcct gggcaacacg gtgaaatccc gtctctacta aaaatacaaa 3840aaaattagcc gggtgtggtg acacatgcct gtagtcccag ctacttggga ggctgaggca 3900ggagaatcac ttgaacctgg gaggtgaagg ttgcagtgag ccaagatggc accacttcac 3960tccagcctgg gaaacagagc aagactctgt ctctgagctg agatggcacc acttcactcc 4020agcctgggaa acagagcaag actctgtctc aaaaaaaaca aaacacacaa acaaaaaaac 4080aggctgggcg cggtggctca cgcctgtaat cccagcactt tgggaggccg aggcgggtgg 4140atcacctgag gtcaggagtt ccagaccagc cttgtcaaca tggtgaaacc tccccccgcc 4200gtctctacta aaaatacaaa aattagccag gcgtggtggc aggagcctgt aatcccagct 4260acttgggagg ctgaggcagg agaatcgctt gtacccagaa ggcagaggtt gcactgagct 4320gagatggcac cattgcactc cagcctgggg gacaagagcg agatttcgtc tttaaaaaac 4380aaaaacaaaa caaaaaacca tgtaactata tgtcttagtc atcttagtca agaatgtaga 4440agtaaagtga taagatatgg aatttccttt aggtcacaaa gagaaaaaga aaaattttaa 4500agagctaaga caaacgcagc aaaatcttta tatttaataa tattctaaac atgggtgatg 4560aacatacggg tattcattat actattctct ccacttttga gtatgtttga aaatttagta 4620aaacaagttt taacacactg tagtctaaca agataaaata tcacactgaa caggaaaaac 4680tggcatggtg tggtggctca cacttgtaat cccagtgctt tgggaggctg agacaggaga 4740gttgcttgag gccaggagtt caagaccgac atggggaatg tagcaagacc ccgtccctac 4800aaaaaacttt gtaaaaattt gccaggtatg gtggtgcata cctgtagtcc cagctactcg 4860ggaggcggag gcagaaggaa tcacttgagc ccaggagttt gaggctgcag tgagctacga 4920tcataccaca gcactccagc gtggacaaca gagtaagacc ctatctcaaa aacaaaacaa 4980aacaaaacaa acaaaaaaaa ccacaagaaa aactgctggc tgatgcagcg gctcatgcct 5040gtaatcccag tattttggga ggcccaggtg ggcgtatcac ctgaggtcag gagttagaga 5100ccagcctggc caacatggtg aaaccccatc tctactaaaa atacaaaatt agccaggcat 5160gtggcacgcg cctgtagtcc cagttactgg gaggctgaag caggaggatc acctgagccc 5220gggaggtgga ggttgcagtg agccgagatc acaccactgc actccagcct gggtgacaca 5280gcaataccct acctcaaaat aaaaaagaaa aagaaaagaa aagttgctgt ccccgctacc 5340ccaatcccaa atccaaacag cctctctcat ctcacagtaa gggggaaaaa tcacccaaaa 5400aagctaagtg atcttttgaa aacccaaact cttagaagtc taagattatt atagtcaact 5460catgaagtgt catcataaaa gatactctaa tattatttaa gtagaaccac atattggttg 5520tcttggtatg tctagcccct ggcatacaaa atatttaata acactgatat ggtacctgtg 5580atgtgaaaat gtactatgag tacagcttta taaatactat atatgtacct atatacagaa 5640aaaaatacaa caaaatcata aaagcactta tctttgaaag aggagttaca gcaattttat 5700ttagttcttt attgctttgc tatatattct aaattttttt caatgaatat atatcacttt 5760taaaaaaatt caatggtctt tcttataaat tatctttggc agcatgcgtt tttatatata 5820catataaaat gtatgggaaa tttttaaagg atacattaaa ttaaagcaaa atatacaaac 5880aaaaaatcag aatacaaaaa gataaaaaga ttgggaaggg agggagggag taaggaggaa 5940gggtgggtgg gtatagagaa atataccaaa taatggtaag aagtggggtc ttgacacttt 6000ctacactttt tttaaataaa aaaaattttt ttctctctct tttttttttt tagagacgaa 6060gtctcgctat gttgcccagg ctggtcttga actcctggga tcaagagatc ctcctgcctc 6120agcctcccaa ggtgcttgga ttacaggtgt gagccaccac gcctggtcac tttctacact 6180ttaatatata tattttttca ttttcaatgt catttttatt agttaattta taatacccat 6240tcaccattat attcaaagtc tatttgaaga aataaaccag aaagaatgaa atactctagc 6300tcacatgcta ttcaatacta aattaccttt caaatcacat tcaagaagct gatgatttaa 6360gctttggcgg tttccaataa atattggtca aaccataatt aaatctcaat atatcagtta 6420gtacctattg agcatctcct tttacaacct aagcattgta ttaggtgctt aaatacaagc 6480agcttgactt ttaatacatt taaaaataca tatttaagac ttaaaatctt atttatggaa 6540ttcagttata ttttgaggtt tccagtgctg agaaatttga ggtttgtgct gtctttcagt 6600ccccaaagct cagttctgag ttctcagact ttggtggaac ttcatgtatt gtcaggttgg 6660cccgtaatac ctgtgggaca acttcagccc ctgtgcacat ggccaggagg ctggttgcaa 6720acattttcag gtaggtggac caggacatgc ccctggtcat ggccaggtgg aggcatagtg 6780ctatacagca ggcagaagtc aatattgatt tgtttttaaa gaaacatgta ctactttcat 6840aagcagaaaa aatttctatt cttgggggaa aagattatgc cagatcctct aggattaaat 6900gctgatgcat ctgctaaacc ttcacatatc agaacatatt tactatagaa agaatgaaaa 6960tgggacattt gtgtgtcacc tatgtgaaca ttccaaaaat attttacaac aactaagtat 7020tttataaatt ttatgaactg aaatttagtt caagttctag gaaaatacaa accttgctag 7080atattataaa aatgatacaa tatatattca tttcaggctc atcagaatat atctgttatc 7140acttgacaag aatgaaaatg caccattttg tagtgcttta aaatcaggaa gatccagagt 7200actaaaaatg acttcttcct tgaagcttac tcaccaactt cctcccagtt actcactgct 7260tctgccacaa gcataaacta ggacccagcc agaactccct tgaaatatac acttgcaacg 7320attactgcat ctatcaaaat ggttcagtgc ctggctacag gttctgcaga tcgactaaga 7380atttgaaaag tcttgtttat ttcaaaggaa gcccatgtga attctgccca gagttcatcc 7440cagatatgca gtctaagaat acagacagat cagcagagat gtattctaaa acaggaattc 7500tggcaatata acaaattgat ttccaatcaa aacagattta cataccatac ttatgtcaag 7560aagttgtttt gttttattgc atcctagatt ttattttttt gatttatggt ttactttaag 7620cataaaaaat ttgtcaatac aactcttccc aaaaggcata aacaaaaatt cataaaactt 7680gcatcacttg agatacttca ggtatgaatt cacaactttg ttacaactta ctatatatat 7740gcacacatat atatatattt gggtatattg ggggggttct aatttaagaa atgcataatt 7800ggctatagac agacagttgt cagaacttgg caatgggtac gtgcaggttc attataccaa 7860gtctacttgt agttgttcaa aatgtatcat aatacaaggc cgggcgaggt cgtcacgcct 7920gtaatcccag cattttggga ggctaaggca ggaggattgc ttgaggtcag gagtttgtga 7980ccagcctggg caacagagca agaccctgtc tccaaaaaga aaaaaaataa ttttttacaa 8040aataaaaaca aaatgtatca tcagacgaaa ttaaataaga ggcaattcat ttaaatgaca 8100acttttccca gcttgacatt taacaaaaag tctaagtcct cttaattcat atttaatgat 8160caaatatcaa atactaattt tttttttttt tttttttttg agacggagtc tcgctctgtc 8220gcccaggctg gagtgcagtg gcgcgatcct ggctcactgc aagctccgcc tcccgggttc 8280acgccattct cctgcctcag cctcccgagt agctgggatt acagacatgc gccaccacgc 8340ccggctaatt ttgtattttt agtagagatg gggtttctcc atgttggtca ggctggtctt 8400gaatttccca cctcaggtga tctgcctgcc tcagcctcac aaagcagtag ctgggactac 8460aggcacccac caccacactt ggttaattct tttgtatttt ttttgtaaag acgggatttc 8520accatgttag ccaggatggt ctcgatctcc tgatctcatg atccgcccgc ctcagcctcc 8580caaagtgctg ggattacagg cgtgagccac cccgcccggc catcaaatac taattcttaa 8640atggtaagga cccactattc agaacctgta tccttatcac taatatgcaa atatttattg 8700aatacttact atgtcatgca tactagagag agttagataa atttgataca gctaccctca 8760cagaacttac agtgtaatag atggcatgac atgtacatga gtaactgtga acagtgttaa 8820attgctattt aaaaaaaaag acggctgggc gctgtggctc atgcctgtaa tcccagcact 8880ttgggaggcc aaggcaagtt gatcgctcga ggtcaagagt tcgagaccag cctggccaac 8940gtggtaaaac cccgtctcta ctaaaaatac aaaaaaaaaa ttagccaggc atggtggcac 9000aggcctgtaa tcccagctac tagggaggct gagacatgga gaactgcttg aatccaggag 9060gcagaggtta cagtgagccg agatcatacc actacactcc agcctgagtg acagagcgag 9120actcctgtct aaaaaaaaaa aaaaaaaaaa agatacaggt taagtgttat ggtagttgaa 9180gagagaactc aaactctgtc tcagaagcct cacttgcatg tggaccactg atatgaaata 9240atataaatag gtataattca ataaatagga acttcagttt taatcatccc aaacaccaaa 9300acttcctatc aaacaggtcc aataaactca atctctataa gagctagaca gaaatctact 9360tggtggccta taatcttatt agcccttact tgtcccatct gatattaatt aaccccatct 9420aatatggatt agttaacaat ccagtggctg ctttgacagg aacagttgga gagagttggg 9480gattgcaaca tattcaatta tacaaaaatg cattcagcat ctaccttgat taaggcagtg 9540tgcaacagaa tttgcaggag agtaaaagaa tgattataaa tttacaaccc ttaaagagct 9600atagctgggc gtggtggctc atgcctgtaa atcccagcac tttgggaggc tgaggcgggt 9660ggatcacctg aggccagaag ttcaagacca gcctagccaa catggcgaaa ccctgtctct 9720acaaaaaata caaaaattag ccgggtgtgg tggcacgtgc ctgtagtccc agttacttgg 9780gaggccgagg caggagaatc gcttgaacct aggaggtgga ggctgcagtg agccgagatt 9840gtgccactgc actccacttc agcctgggcg acaagagcaa gactccgtca caaaaaaaaa 9900aaaaaaaaaa aagcttaaaa tctagtggga aaggcatata tacatacaac taactgtata 9960gcataataaa gctcataatc tgtaacaaaa tctaattcga caagcccaga aacttgtgat 10020ttaccaaaaa cagttatata tacacaaaaa gtaaacctag aacccaaagt tacccagcac 10080caatgattct ctccctaagc agtatcaagt ttaaagcagt gattacattc tactgcctag 10140attgtaaact gagtaaagga gaccagcacc tttctgctac tgaactagca cagccgtgta 10200aaccaacaag gcaatggcag tgcccaactt tctgtatgaa tataagttac atctgtttta 10260ttatttgtga cttggtgttg catgtggtta ttatcaacac cttctgaaag aacaactacc 10320tgctcaggct gccataacaa aataccacag actgagtgac ttaacagaaa cttatttctc 10380acagttttgg aggctgggaa gtccaaaatt aaggtacctg caaggtaggt ttcaatctca 10440ggcctcttct ttggcttgaa ggtcttctaa ctgtgtgctc acatgacctc ttctaacaag 10500ctctctggtg tctctttttt tttttttttc ttttttgaga cagagtctca ctctgtcacc 10560caggctggag tacagtggca caatctgggc tcactgcaac ctccaactcc cgggttcaag 10620tgattctcat gcctcaccct cccgagtagc ttggatgaca ggagcccgct accacaccca 10680gctaattttt gtatttttag tagagatggt gtttcactac attggccagg ctggtctcaa 10740actcctgacc tcgtgatcca cccaccttgg cctcccaaag tgctgggatt acaggtgtga 10800gccactgcgc ccgtcctggt gtcttttcat ataagggcac taatccaatc agacctgggc 10860ccaaccctcc cgacttcttc taactgtaat taccttccaa aggccctgtc tccaaatacc 10920atcacactgg gggttaggac ttcaaaaaag gtatgggggg ggtgtgggag gacataaatg 10980ctcagtccat aacaagcacc caacataaaa atggctagaa cagatcacaa aaaaaaggtc 11040ctgtatggct ttggggaagg gctcaacccc aaaatatctg agagctctgg aggggcctag 11100aagtggtaaa tgaatgaaaa cgtggttact ctccagatct gcctttccca aatatggcca 11160ttcttggctg aatcagaaat caaaggacag gttattaatt actagctcta agttacttac 11220catttgctga gacagttcag aaatctgact gcatctcctc agagatctag aacacagttc 11280tcaaattcta acttacttgt gatatacttg tgaatgataa aaatcgctac aggtactttt 11340attaatctga aagagtattg agaaattacc tttcattctg acttttgtct ggaatgaaaa 11400tcaatacttt tgctataatc gattactgaa ataattttac tttccagtaa aactggcatt 11460ataatttttt ttaattttta aaacttcata attttttgcc agactgaccc atgtaaacat 11520acaaattact aataattatg cacgtcacat ctgtaataat ggccttcatg taaacatttt 11580tgtggtttac acataaaatc tctaattaca aagctatatt atctaaaatt acagtaagca 11640agaaaattaa tccaagctaa gacaatactt gcaacatcaa ttcatcatct gtgacaagga 11700ctgcttaagt ctctttgtgg ttaaaaagga aaaaaaaaaa aaagacatgt tggccagatg 11760cggtggctca cacctgtaat cccagcactt tgggaggctg aggtgggcgg atcacccctg 11820gcctgcccaa catggtgaaa ccccgtctct actaaaaaca caaaaattag ctgggcgtgg 11880tggcgggcgc ctgtaattcc agctactcgg gaggctgagg caggagaatt gctagaaccc 11940aggaggcaga gattgcagtg agctgagatt gcaccattgc actacagtct gggcaacaaa 12000agtgaaactc catcttaaaa aaaaaaagac aatgttcgtg ggtccaaaca agacttaatg 12060gaagtgagtc taaaaatgag ctatgtgggc caggcgtagt ggctcccacc tgtaatccca 12120gcactttggg aggccgaagc aggcagatca tgaggtcagg agatggagac catcctggcc 12180aacacggtga aatcctgtct ctacaaaaat tagctgggcg tggtggtgcc tgcctgtaat 12240cccagctact cagaaggctc aggcaggaga atcgcttgaa ccagggagtc ggtggctaga 12300gtgagccgag atttgcatca ctgcactcct gcctggtgac agagcaagac tccatctcaa 12360aaaaaacaaa caaaaataaa agataaaaat gagctatgtg aattaaaaga ggtataacaa 12420tagataaacc atattttatt taattcctag taatgagtaa tatttccaaa cttctggaat 12480gggcagaaat tgctagttgg catattttta ccttttatat tcagatacat taaaattctc 12540aaaaaaaaac acctcaaagc agatgatccg ccatctcctt ggataatttg tgttaactca 12600ggataacaga aaaccaaaat tatgagttac tgatgcaata ttcctaaatg taaaaataat 12660taaagctaat agtagattca tcttccaatt tcatatcagt cttacaaata aactacatat 12720ataacttgct tgccttccct tctgagggat aaagctgtta gaagaattaa aatcagcatt 12780cttgactatt caaccaaggg agggataaat tattactcat tctagggaca tgggctcata 12840actactacat gtgtaaggac atgaatttac ccaatattac aatttttcct tttattagtg 12900tgtacagtgg aagaatagac atgttcactc tggacaaaaa aaaaattata cttatcagtt 12960atcagaagca caatgctgaa gacagtagtt ccataacaat ttgaagtatg tgatcgaact 13020agtagattat cttagtagta gtgaattatt gtaaatgtta gtaatttggc agccactggg 13080cagaaaaata agaattgagg ctcaatattg atattaatgg tggtgattga cacataaatt 13140ttatcaagtc tacacaatat aaaattacag aaaggtagaa gagtatacca gtacaacttc 13200aacatatctt cactacaagg gagtaaaatg acatggccta gttactatct aatgaactgc 13260agaaaactaa aagaaaactc caaggcaact cttctctgct gatctggttg gtccttttcc 13320taccttttgc aatacccaga tacaaacaat ggatagaaaa caaagtagac ttgtagtatg 13380caggtcacag tgctaaattc acagaaagaa acccctgaac tgaactgctc tatttcctgg 13440tggtcacaaa gagtaattct ggtttacacc tacagattga tgtcaatcta caccctgttg 13500ataacagtgt ggccaaggac aaaaaaaagg tgctccgttt taccaattct gtaaaaaatt 13560attggcaggg taagctcggc tagggcagga ttacatttct aggactacca tccccgaaat 13620ttagaagata ttatatccac ataaagcata tctttcacat taatttgcaa aaatctaaaa 13680gctttttctt agctcaagtg tgtccaagtt taccctggca gtttaaaacg atagttacaa 13740gcagcatggg ttgtatcaga cacatttgag ggccaatttc atgtaagtga tattgggcaa 13800gttacttcaa ctatctgtgc ctccaaggtc atactagtgt ttatttacct aaagggtacc 13860tgttatgtaa ctttagggtg tttacattag ataatgcctg caaaatattt acttcaacgc 13920ctaaaacata gttaagtatt caataaatac ctactattgt cactactaac ttaaaagttt 13980agagattaag agcagaatct ggggtgagac aaacttaggt tcaaatccta gtattgttgg 14040gtaatcttgg gcaagttact taacctctct gatttgtgta atttaaaaaa ttagttaata 14100tacataacag ggcttagaag agtatctagc acatagcacc atttaagcat ttgttattgc 14160taacatgcaa acaatttaag ggaaagaaat tttttaaaaa ggaagaggga tttgcaaact 14220aaaaacaatg agtatcttat gttcaaagaa aactaacaaa cagccagctc tagcaataat 14280taaattcact atatactggg gcaggcatca caccccaaag ctaaaagcgt ctacctaggc 14340caggcacggt ggctcatgcc tgtaatccca gcactttggg aagcagaggc gggcagatcg

14400cttgagctca ggagttcaag accagcctgg acaacatggc aaaacaccat ctctacaaaa 14460aatacaaata ttaggccggg cgcagtggct cacgcctgta atcccagcac tttgggaggc 14520caaggcgggt ggatcacctg agatcaggag ttcgagagta gcctggccaa catggtgaaa 14580cctcgtctct attaaaaata caaaaaatta gccaggcatg gtggcaggcg cctgtaatcc 14640cagctactca gggggatgag gtaggagaat cgcttgaacc cgggaggcag aggttgcact 14700gagccgagat catgccactg tactccagcc cgggcaacaa gagcgaaact ccatctcaaa 14760aaataaataa ataaataaat aaaataaagt acaaatatta gccagggatg gtggtgcgca 14820cctgtagtcc cagctacttg ggaggctgaa gtgggagaat cccctgagcc tggggagaat 14880cacccgagcc cgggaagtcg aggctgcagt gagcagtgat tgtgccactg cactccatcc 14940taggtgacag agtgagaccc tgtctcaaaa aaaagaaatt ggcagaatta agtaagttga 15000tgtttagaga tgaaaaatca acattttttc ctcagcaact gaataaaaac aacagccact 15060accatttttt tgagtaccta tttgtagcct attttttaac tggtattact cgagagagag 15120agagctaggt tcgagacaga gctccttctc ttaataactg tatgacctag ggtatgtctg 15180ttagcctctc tgaggcttca aaggttcctc atctgtaaaa tggtaataat cataccattg 15240ctacagggct gttttgaaga ctaattagga ctatgtaagt aaacatgatg atggctatta 15300ttactgttcc ccgccagggg ccatgcaagg gttgctgatt cacatagact gtcttataat 15360cctctcaata actccaagag gtagccagca cctcagatat acataaaatg acttaagccc 15420agagaggtga agtaagttgc ccacagccac acaactagta aatagcccaa acaagctgga 15480ttcccagtta gactccgtta atagcactgc tctttacctt aagtcattac aatgcctaat 15540atgaaataga atcgcttctt tcttagggtt caagtggtta attatttaat gtattcattc 15600aacaaaccat catcgaggac ctcttacaag ccaagtactg tgctaagtgc tagagttacg 15660gcggtgattc ctgcccttaa aaagttttag tgggagaaac aacaggtaac caggtcattg 15720ccaaaacaac aaaaataatc ataataaagc aggctaaagc atatttaact ggccggggtt 15780ttgactattt tagcaagcat gatcagaacg gttgaggagg gaggccagca gcttggccgg 15840ttcaacaaac aagaaaaaac cagtgagggt ggagctaaga taccagaggc tgattacggt 15900taagaatgtt cttgaaggta aggaccagat tctcattttc tatatcctgg ggcatcggtc 15960agcatggaat ctggattcta gcacatgtga atttcggctt gaaatgacct aatgcctttt 16020ccctagttcc ttcgtgtgtc aaatacgcat ggttaccgct accagagctg tagtggggct 16080tcaatgaggc catgagcatc tccataaaga tgaactacag tgtgtgcaaa actaaaggca 16140aaacctggtc cccacacgcc ctcccaggtg gtcgctttcc gtgccgaggc ccctccagag 16200gtgccccgag aacctcacca tcgcacccca aacttccagg gaagggcctc tcccgagaaa 16260gcccccacgc ccccaccccg cgccatcatt cccgaatctg ccctcggccc ctccccgcag 16320cacgctcgca ggcggcacat gtcaaccaaa acgccatttc caccttctct tcccacacgc 16380agtcctcttt tcccagggct cccccgagga gggacccacc ccaaaccccg ccattccgtc 16440ctccctgccg ccctcgcgtg acgtaaagcc gaacccggga aactggccgc ccccgcctgc 16500ggggttccct gggcccggcc gctctagaac tagtggatcc caattgaagg cctggtctaa 16560atgactccaa aatcaccact taattcaaga gactgatttc cctgagtcag gccccttaaa 16620gcagctattt caatgggaca gggaaacaac cctaggatct ggattagaat cacttggggg 16680ctgccacacc cccagggctc tgatcctgcc cttctcccac acgcacattc acatactgct 16740gcagtgacct tccatttcta atgggttcct gggccatctg tcaggtatag ggaatggaaa 16800aggggttggg gaggctctgc ttcagaaagt ttgtgtcagg ggctcccaga gcctccacag 16860atagatagca ggggtcccca ccctaccatg gcagctataa atgtgatcaa catttattgg 16920cctaggatac agcagttagc aaaatgcctg atgtagttcc cactccgtgg aggttgcagg 16980ctagccaaga agtcatgagt tcagcaaccc ttacgcacca gtgggatgag attggaccag 17040gccgagggta gtcttgggaa cactcagcat ttgtctgagg gccagaagag gctgcttgcc 17100ctcagacagg aggtcagcat ctttattgta gcccatgaca cctctacacc attgctcttc 17160tggtcttatg gaagacatct ttgggcctga taacagcgga gtctgtgtcc cacttgtcca 17220ggctggagtg ccacatcagg cacactccag ttgcagggac agcacagaca agtttcagga 17280aggctggtgg cctccaggag gttaacctta taaggccaga ttgtaaccta gttgaaaaac 17340atacacatgc catgataata aaagaaccta ggcaccatta caagagaaaa aatcattttt 17400gtagatacga gcatggattc ttgggtgggt cagacacact gggcttgtgc tctgactgca 17460ctgtctcccc tacctgacct tgggtaaacc ataagactgc tgcatgactc agtgtccacc 17520ccaaaaaagt accggtagat attggccaca gtagatatca gctagagtgg actctcatga 17580caatgagggg agatgtattc cccatcttag gcacctggga ctctaccttc catcttctgc 17640tccgtgtctc tccatcccca ggctcttcag aactcaggga gtccagaatg tcagctccca 17700gatttcagcc ttcagaaagg aaacccatta ccgttcagtt gaacaaatgt tgtctgagcc 17760ccagatctgg gctcagaggc catctaggct atgagacaag aggggaacaa agcaccgtct 17820gcactcactc accacactca cttgctgtcc caggtcacat ccatcgggta gagaatctaa 17880gaggctgagc tagctcccgc caccagccca gcccacccca cctggcccct tccttccttc 17940tacaaaatat gcaccacctg tcaaagggtg ggcagtgcca ggcctgcata cagagcactg 18000agtgtaaaag cagacatgga ccctgacctc caggagcttc caattttctt gaagagacaa 18060atcagctggc atttcagtcc agtgtgatct gctcttggtg agcacagacc tagggagttg 18120gggcagcttc ccagaagaac tgcagtccag gctgagggca gagaaatgag gggaatggcg 18180aggaattggg gagcaggggg gagctcagta gagagccaag ggcgggaggt gagaagtccg 18240tgttgggcca ggagctaccc tccggtggcc acagccgaag tcgaggatgc ctttggaact 18300catccccact tctctctttc tgtatgtagc cgtccaagaa caagtcacct ccaagtgtag 18360ccggatcaag gcaagccccc catctagcaa gcacttgatg ccacccagaa ctgggcttct 18420tcagaacaat ctgagtccag gaatgatccc actcaccagg caccagagct gcgagggcat 18480gggagtgatc tcaccaactc tggggaagcg gcaaggaatt ttcacctcca gcccccagtg 18540tcccatcctc tcacactcag gccagactcc cctgggcaga cttgactctg tctgccagca 18600tatgcagagc cccaaggcca ccccaccaga agtgcccctg cctgggttct gtcccagctc 18660cctgggcacc cagtccttga gtccccacca gctcagacgg cctagtgtgc caagaatgcc 18720cactgcgttc aacaatgctg catgggtcac agcggcagca gctgtgacca cagcagtttc 18780ggggaaaaca cccctcagcc aagtggataa tagcgttcag cagcactcac cttctggcca 18840ggcctgcctt cagaggccat ctgattggga ggcacaagtg cccgctgcga tgggaacaca 18900agtgcccctg gccaacaacc ccagcttcag cctgctgggc agccagagcc tcaggcagag 18960cccggtacag ggcccggtgc ctgtagcaaa caccaccaag ttcctccagc agggtatggc 19020cagctttagt cccctgagcc ccatacaggg catcgagcca ccaagctatg tggctgctgc 19080tgccaccgct gctgctgctt ctgccgttgc tgccagccag ttcccaggtc cgttcgacag 19140aacggatatt ccccctgagc tgccacctgc cgactttttg cgccagcccc aacccccact 19200aaatgatctg atttcgtcac ctgactgcaa tgaggtagat ttcattgaag ctctcttgaa 19260aggctcctgt gtgagcccag atgaagactg ggtgtgcaac ttgaggctga tcgacgacat 19320tttggaacag catgctgctg ctcaaaatgc cacagcccag aattctgggc aagtcaccca 19380ggatgctggg gcactttaaa tctgagcagg atgcccatag aaacccccat ggtgacatca 19440ctctaggaag tggtgtcgat ccatacccgc agttgtctcc cgttacaatt tgagtggtgt 19500tgtcagccca tgcttatccc tctctctacc tgtgacaaaa tggaaagctg gtgatttttc 19560aagctacgtg tacatatttg aaaattttgt aaatggtttt cctaaacatt aatgacagaa 19620gtatttatac ttcattttgt gactttgtaa ataaagcgac ggcttttgtt tcagtagagt 19680tgtgtttact atgcattgtt ttgtgtttat tatacaatgt tacaaatatg cagaccgtgt 19740tgtttgctcc agtgatacct tgttaagcta ggtggctgag tcgcttatgg ttttaatgca 19800atgagcaatg tggatatgac caagagttgt tgtgcaagtt gacaaatgcc aaatagaaaa 19860ccacttggcc atttatttct atgttcacta aaaatcctat tgccttgtgt gattcttaat 19920ctcttttgcg aacctttcag tctccgctag ctctttccta atgagcttta cagcagaagc 19980tgttttatcg ttaagtgccc cacagagaca ctttaccagg aggctgggag agttctccag 20040atttgggaga ggcgcagaga cagtgtgtga gccgagccct gtctcagcaa tccacctgga 20100ggagctagag tatcctcctc cctttaccat tcagaccgag agaaaaagcc cagcttgtgt 20160gcaccctcgt ggggttaagg cgagctgttc ctggtttaaa gcctttcagt atttgttttg 20220atgtaaggct ctgtggtttg ggggggaaca tctgtaaaca ttattagttg atttggggtt 20280tgtctttgat ggtttctatc tgcaattatc gtcatgtata tttaagtgtc tgttatagaa 20340aacccacacc cactgtcctg taaacttttc tcagtgtcca gactttctgt aatcacattt 20400taattgccac ctcgtatttc acctctacat ttgaaatctg gcgtctgttt caagccagtg 20460tgttttttct tcgttctgta ataaacagcc aggagaaaag tgcctctatg tttttatttt 20520tcaagggagt attcagtacc tacaaaccca agtcaggaag cctgctagtg gctttggttc 20580tttcagaggc tgctcgatgc cttgtgtgtc agaaagaaag attcagcagt tttgcatcat 20640ggcaaagaag cctgttattt tggggctcag cccctcattt tatagaggat gaaacagagg 20700gggatgggag gtcacaaaga caactgcccc gggagcaggt gtgggggaga cttgccctga 20760gggtctagac gctctgcacc accgtcctgt ctcccttgct gaagaccaca catgcccttc 20820tttgaccaga ccctgccacc tgataggcca ggacctggta ggcgggtacc caggtttcat 20880ggatggaacc acatctcccc aaaagtgggg aggtagctac tgggatgcac gcctcccgcc 20940atgtgctata ggagagcagc tgaagcaaca gttgggatca gatgtagtca caattgaatg 21000catcatcaca tttatccctc taagtggctg ggagagttga tatcctcatc cctaaggtac 21060aaaatgttcc aatttgatca gtggctttca ggagctgaga aaggcatgtg ctctgaggca 21120gagctgttat gtcccgcaga gcctaaaaat gctctaagaa catgctccct gccaaaattc 21180tcaatggctg tgacaaggga caacgatcga ccaatggggg tggaagcaga cctccgcagt 21240ccaggggcca gagctaggac agaggggtcg gagaaagagt cattttccca acactccagc 21300tcttggccag tcctcacaca gtcccctcct gcttcctgct gagagagata tcctcatagg 21360tctgggtaaa gtccttcagt cagctttcat tccctgtcac caactttgtc tctgttctcc 21420ctgcccgtct caggcagcac tcctcaggaa acctctccaa gagccagcct cactgcagcg 21480cccactattg tccctctgcc tcaagtgtcc catccatgcc aggccccagg caggctgcag 21540ctttccctca gggccacacc aaagcacttg ggctcagctg tgctgtcccc ctccatcact 21600gagctcaggg gcagcagggg tggggtgcca ggaggcccat tcacccttct ctggctctgt 21660gttggaccca cctgcccagc cactgctgct tagaacctac ccgctgggaa aatgaagccc 21720tcccggaggg gccacctcaa cctgagagcc tcacggatca cagttgtccc cactcagctc 21780tgccagccct cagagaccca tagataaaag ctgagcttgg ctcgcagagc tggttccatc 21840ttccattccc agagggttca acttcctacc ccaaccacac agggaacctc aaggctgagc 21900cagtgtgggc tgcagtgcag accagcttcc tggacacgtc ctgccacctg accccaggct 21960ggcctcactg cccctggcac tcctgaccct atcctcattc ctcctggcag tgcgtgttct 22020gccattccgc tttcccttag ctgtcctctc actgtactgt cagcttctcc ttttccaggt 22080gccccccagg ggctttccac atgaccctgt caccccacag cccatccagc accaattcca 22140gctctctgcc acccttcaaa ggagtgacag tgccctgctt cacctcccac tcacccctca 22200acccagagca atctggctcc agtcttgcct ccttccccct aagtactcta gtcacagttc 22260caaattcctc ctggtcataa agccaaatga agcttcctgg tcctcagcgg acttgccact 22320tcagcagtac tggactctct cctcccagaa acctgtttcc ccttggctcc tggagcccac 22380actctgctgg aatccttctg cctctctggc ctgtagcctg gccctctctc ccaacctgag 22440gtccattctc tcctgctcct ccacaagatg ttgctccttc cattacttcc tccctctcaa 22500ccaaagctcc ttcattagct ctttatcttc tggtttcttc ccctgggcag acgaatggat 22560tcaagagcct gtggcccagc agcccagcac tccaggatct cagcacttca gcatcccagt 22620accctagcat ctcaataccc cagcacccca gcaccatagt attccagcac cccattgtcc 22680aagcatctca gcactccagc atcccagcac cccaacactc cagcagccca gaatctcagc 22740accctagcac tgcagcatct caggacccca gcacttcagc atcccagcac actagtactc 22800cagcatctcg gcaccccagc acctaggcat cccaacaccc agcaccccag cacttaagca 22860tcccaccact acagtatctc aacactccag caccccagca ccatagtgtt ccagcacccc 22920agcatcccaa caccccagca cttaagcatc ccaacacctc ggcatcccaa caccccagca 22980ctgcagcatc tcagcacctt agcatcccag tgccctagca tctcaatgct ccagcacacc 23040agtactacag tattccagca ccccagcact ccagcatctc agcactgcag cactgcagca 23100ctccagcatc ccaaaatccc agcatcccaa caccccagca gaccagcaga ccagcatctc 23160agcaccgcag catccaagga ctatcccagc atcccagcaa cccagcacct cagcatccca 23220acaccccagc atttcagcat ggcaacaccc cagtacccca gcacttcagc accccagtat 23280cccagcatct cagcgaccca gtatcacaaa acctcagcat cctagcaccc cagcacccca 23340gcaccttagc accttagcat cccagcatct cagcgcctca gcatcttgat attctggctg 23400aggtcagcgt ggtgtatcta gtcagggtcc taactttcac ttcgcaggga aatgctgctg 23460gactgggtct catgttgggc tgaagctctc tagacccctt gaagacagca taaaagagct 23520tggagacgct gggtgtcccc catggaagag ttcactctca tcctgctttg acaacagcct 23580tctctggggt ccctcacggg cccctctttc ttactgcaag tttgtctctg agaagactgt 23640gatgcagaag tcactcagct gcctgtggct cctgaagagc tgaaggtgga ggcctgtagg 23700cctccctatg agaggcgcag aaaaaaccat gattgctagt ggggaggtgc tccctctaca 23760acccactcca taatctgccc ccgcccagct ctgaggccag ccccagggga aaatgccaga 23820tccccaggga ggtgtgtgag acctcagggg ctccctcctc ccttacagca ggctcaggcc 23880cctgggggcc tcagggccaa ggtctgtggg taagctacta tctctcactt gtcctctagc 23940cacaaaagcc agggagatct ggcaatggac atgaggttct gaagaagcac atatgactgg 24000cttcctaatg cgtggttgtt cagtgattca ataaacacgc atgggccagg catggggaaa 24060tagacaaaca tgatccccaa cctctcccag agtgaactgg gagggaggag tgttcatccc 24120tcaggattac accagagaaa caaaccagca ggagatatat atggttttgg ggggtcaaga 24180aagaggaaaa acctggcaag gcaagtccaa aatcatagga caggctgtca ggaagggcag 24240cctggaacct ctcaagcagg agctgatgct gcagtccaca ggcagaattt cttcttcctc 24300ggggaaatct cagctttgtt cttaaggcct ttcaactgat tggctgaggt ctgccccttc 24360ccccacattc tccaggataa tcttccttac ttaaagtcaa ctattaatca cagctacaaa 24420atcccttcac agctacacat agatcagtgt ttgattgacg aacagcccct acagcctagc 24480caagttgaca cataaaacta accatcacag ggggacaaat gatgtaaaca catcaacaaa 24540taaaacagta acaagttaag gtctatggaa aaaacacaga aggggcagag agaaagaaag 24600caagaaggag agtcccagtt tgctagggct tgtgggaagt ggggagcagt tctctttagc 24660taggatattt gggaaaggca tatctgaagg agtgatattt gagcttagat taaaagatgg 24720gaaggagcaa gccatgcaaa gagctaggat gttccaagca gagacggaac agcaagtgca 24780aatgtcagga ggaatagaag gaggctggtg ggtggggtcc agtgagcaag aggagggcag 24840gcaggagagg ggatggggag gtgggcaggc ccagaccacc cagggccctg gagactatcc 24900tgatccaaca agggaagcct tgagtcactt cagtgtccat gtggagaatg gacctcagac 24960tgaatgaggg aggcagtaag gagggcctct acctccaggg cttcgccctg tggactgcgc 25020atagacatct ccaactcaga aagtctgaac caaactttcc atagttcccc caagtctggg 25080catcctccta ctcagtgaaa ggcagccatc acacctccct gccctgctcc cggatgcccc 25140aaatcctctt ggtctccaag tccagaacct gagacttgtc cttgatgttt gtctttccct 25200caccctttct gtattctggg aagatgggtt tttttccccc agatgaatct gtaaaacttc 25260tgtgatcaca ataaaaattc tggcagtatt attttctgga acatgacaaa gtgattcaaa 25320attatttatc tggaagacta caaaacaaga atagccagga aatttctaaa aagaaagaag 25380aaggaggagg agaaagaagg aggaggaaaa ggaggagaag aagaaaagaa aaagaaccaa 25440gaaagggttc tagctctacc aaatattaaa acatatcatg aagctattta aaacaatatg 25500gttgtggata ctgaaaaaga tgtgaataaa gtggaaggaa aataaataga aatgcacatg 25560gggattgaga ctgtgaaaaa ggcagcatct cacatcagtg agggatgttc aacacctggt 25620gttgggaaaa ctggctagtc atttaaacca aacaactggg tcctctacct cactcctgac 25680attaagatac atttagatga ttcaaagagt aagacagaaa aaataacacg tgaaaacact 25740atcagaaaac aacgtgggcc aggtgtggtg ggtcacgcct gtaatcccag cactttggga 25800ggccgaggca gacagatcac ctgaggtggg gagttcaaga ccagcctgac caacatggtg 25860aaatcctgtc tctactaaaa atacaaaatt agctgagcgt ggtggcgcat gcctgtaatc 25920ccagctactc aggaggccga ggcaggagaa tcacttgaac ctgggaggca gaggttgtgg 25980tgagccgaga tcacgccatt gcactccagc ctgggcaaca agagtgaaaa tccatctaaa 26040aaaaaaaaaa aaagccaagg tggatatttt tatagtatca gggtagatca agcttctcca 26100atcatgacat gaaacccaga aaccataaaa gaaaagaatg ataaaattgc ccacgtaaag 26160taaaaagctt gcacacagaa aaacaccata caggttacaa gatgagcagc aaaatcagag 26220aaaaaacatt gcaattcagg acacacagag gctattgttc ctaatattta aaaataaaag 26280tagtggattg tctacaaaaa gatgaagaca agaatttcag aaaaccaaat actgcatgtt 26340ttcacttaca agtggaagct aaacactgag tacacgtgta cacaaagaat ggaaccatag 26400gccaggcacc gtggctcacg cctgtaatcc cagtactttg cgaggccgaa gcgggcggat 26460cacctgaggt gaggagttcg agaccatcct ggccaacatg gtgaaaccca gtctctacta 26520aaaatacaaa aattagccgg gcgtggtggt gggtgcctgt aatcccagct actcgggagg 26580ctgcggcagt agaatcgctt gaaccctgga ggtggacctt gcagtgagcc gagatcgcac 26640cactgcactc cagcctgggc aacagagtga gactccatct caaaaaaaaa aaaaaggaat 26700agaacaatag acactggggc ctacttgagg gaggagggtg aggatcaaaa acctgcctat 26760caggtactat gcttattacc tgggtggtga aataatctgt acaccaaacc ccagtgacat 26820gcaatttacc gatgtaacaa acctgcccat gtacccgctg aacctaaaat aaaagttgga 26880aaaaaatata gaaattttct ttgtaatagc caaaaactgc aaacagccca ggtgtctatt 26940agtagaatgc ataaacaaac tcgggcatgt tcatacaatg taaaactact catcaataaa 27000aagtgatact tctcagcaat gaaaagaaac tagctactga taccagctac aacatggatg 27060gatttcaagt gctttatgat gagagcaaga agccagacac aaaagtgtct atatatatat 27120acagtatata tacgtatata tacacatata tacagtatat atatacatat acatgtatat 27180atatactgta tatatactgt atatatatac acagtatata tatacatata tacagtgtat 27240atatactgtg tatatataca tgtatatata ctgtgtatat atacatgtat atatactgtg 27300tatatataca tgtatatata ctgtgtatat atacatgtat atatatgtat actgtatata 27360tactgtatat atatatacac atatatacag tatatatata cagtatatac tgtatatata 27420cagtatatac gtgtatatat acatatatac agtatatatg taaatataca tatatacagt 27480atatatgtaa atatacatat atacatgtat atatatacac tatatatata catatatagt 27540gtatatatac atatatacat gtatatattt actatatgat tccatttata taaagtgcca 27600aaacagtcaa aaataatcta tgtggaaaaa atcaacaaag ggatcccccg ggctgcagga 27660attcgatggc gcgccgacgt cgcatgctcc tctagactcg aggaattcgg taccccgggt 27720tcgaaatcga taagcttgga tcggccgcaa taaaatatct ttattttcat tacatctgtg 27780tgttggtttt ttgtgtgaat cgtaactaac atacgctctc catcaaaaca aaacgaaaca 27840aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa catttctcta 27900tcgaggatct gcgatcgctg aattctgggg actttccact ggggactttc cactggggac 27960tttccactgg ggactttcca ctggggactt tccactcctg cagcagtgga tattcccaga 28020aaactttttg gatgcagttg gggatttcct ctttactgga tgtggacaat atcctcctat 28080tattcacagg aagcaatccc tcctataaaa gggcctcaga ggaagtagtg ttcagctgtt 28140cttggctgac ttcacatcaa agctcctata ctgacctgag acagagccat gaattccgtt 28200tttttttttt tttttggaaa tatgagggtt tttccgcttc tgacagtgga acggaatgac 28260agcagcacag gctggtgaat gactactttc tttataagca accaccttga gcctgaaatg 28320gcagtcgcta gtctctattg ccttgctgtg gcctcgggat ggaaatctgc tggggaccct 28380acagtcacct aatctctctc cttctcatcc ttctgtttca ttcagaggca gcctgccgcc 28440cttctgggaa aagaccctgc aagatgcaag ccttcagaat ctgggatact aaccagaaga 28500ccttttacct gagaaacaac cagctcattg ctgggtactt acaaggacca aatatcaaac 28560tagaagaaaa gatagacatg gtgcctattg accttcatag tgtgttcttg ggcatccacg 28620ggggcaagct gtgcctgtct tgtgccaagt ctggagatga tatcaagctc cagctggagg 28680aagttaacat cactgatctg agcaagaaca aagaagaaga caagcgcttt accttcatcc 28740gctctgagaa aggccccacc accagctttg agtcagctgc ctgtccagga tggttcctct 28800gcacaacact agaggctgac cgtcctgtga gcctcaccaa cacaccggaa gagcccctta 28860tagtcacgaa gttctacttc caggaagacc aatagtctag ctcgacatga taagatacat 28920tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat 28980ttgtgatgct attgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt 29040ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag 29100ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tagatccatt 29160taaatgttag atccggagag ctcccaacgc gttgggtcga cggcgcgcct accagtaaaa 29220aagaaaacct attaaaaaaa caccactcga cacggcacca gctcaatcag tcacagtgta 29280aaaaagggcc aagtgcagag cgagtatata taggactaaa aaatgacgta acggttaaag 29340tccacaaaaa acacccagaa aaccgcacgc gaacctacgc ccagaaacga aagccaaaaa 29400acccacaact tcctcaaatc gtcacttccg ttttcccacg ttacgtcact tcccatttta

29460agaaaactac aattcccaac acatacaagt tactccgccc taaaacctac gtcacccgcc 29520ccgttcccac gccccgcgcc acgtcacaaa ctccaccccc tcattatcat attggcttca 29580atccaaaata aggtatatta ttgatgatgt tt 29612329273DNAArtificial Sequencehelper-dependent adenoviral vector-equine Il-1Ra gene 3aaacatcatc aataatatac cttattttgg attgaagcca atatgataat gagggggtgg 60agtttgtgac gtggcgcggg gcgtgggaac ggggcgggtg acgtagtagt gtggcggaag 120tgtgatgttg caagtgtggc ggaacacatg taagcgacgg atgtggcaaa agtgacgttt 180ttggtgtgcg ccggtgtaca caggaagtga caattttcgc gcggttttag gcggatgttg 240tagtaaattt gggcgtaacc gagtaagatt tggccatttt cgcgggaaaa ctgaataaga 300ggaagtgaaa tctgaataat tttgtgttac tcatagcgcg taatatttgt ctagggccgc 360ggggactttg accgtttacg tggagactcg cccaggtgtt tttctcaggt gttttccgcg 420ttccgggtca aagttggcgt tttgatatca agcttatcga taccgtaaac aagtctttaa 480ttcaagcaag actttaacaa gttaaaagga gcttatgggt aggaagtagt gttatgatgt 540atgggcataa agggttttaa tgggatagtg aaaatgtcta taataatact taaatggctg 600cccaatcacc tacaggattg atgtaaacat ggaaaaggtc aaaaacttgg gtcactaaaa 660tagatgatta atggagagga tgaggttgat agttaaatgt agataagtgg tcttattctc 720aataaaaatg tgaacataag gcgagtttct acaaagatgg acaggactca ttcatgaaac 780agcaaaaact ggacatttgt tctaatcttt gaagagtatg aaaaattcct attttaaagg 840taaaacagta actcacagga aataccaacc caacataaaa tcagaaacaa tagtctaaag 900taataaaaat caaacgtttg cacgatcaaa ttatgaatga aattcactac taaaattcac 960actgattttg tttcatccac agtgtcaatg ttgtgatgca tttcaattgt gtgacacagg 1020cagactgtgg atcaaaagtg gtttctggtg cgacttactc tcttgagtat acctgcagtc 1080ccctttctta agtgtgttaa aaaaaaaggg ggatttcttc aattcgccaa tactctagct 1140ctccatgtgc tttctaggaa acaagtgtta acccacctta tttgtcaaac ctagctccaa 1200aggacttttg actccccaca aaccgatgta gctcaagaga gggtatctgt caccagtatg 1260tatagtgaaa aaagtatccc aagtcccaac agcaattcct aaaaggagtt tatttaaaaa 1320accacacaca cctgtaaaat aagtatatat cctccaaggt gactagtttt aaaaaaacag 1380tattggcttt gatgtaaagt actagtgaat atgttagaaa aatctcactg taaccaagtg 1440aaatgaaagc aagtatggtt tgcagagatt caaagaaaat ataagaaaac ctactgttgc 1500cactaaaaag aatcatatat taaatatact cacacaatag ctcttcagtc tgataaaatc 1560tacagtcata ggaatggatc tatcactatt tctattcagt gctttgatgt aatccagcag 1620gtcagcaaag aatttatagc cccccttgag cacacagagg gctacaatgt gatggcctcc 1680catctccttc atcacatctc gagcaagacg ttcagtccta cagaaataaa atcaggaatt 1740taatagaaag tttcatacat taaactttat aacaaacacc tcttagtcat taaacttcca 1800caccaacctg ggcaatatag tgagacccca tgcctgcaaa aaaaaaaaaa ttagccaggc 1860atggtagcat gtacctgtag tcccagctac ttgagaggtg aggtgggaaa atcactttag 1920tgcaggatgt tgaggctgga gtgaactgtg attgtgccac tgcactccag cctggacaat 1980agagcaagac cttgtctcaa aaaaatgcat taaaaatttt ttttaaatct tccacgtatc 2040acatcctttg ccctcatgtt tcataaggta aaaaatttga taccttcaaa aaaaccaagc 2100ataccactat cataattttt tttaaatgca aataaaaaca agataccatt ttcacctatc 2160agactggcag gttctgatta aatgaaattt tctggataat atacaatatt aagagagact 2220gtagaaactg ggccagtggc tcatgcctgt aatcccagca ctttgggagg ctgggtaaca 2280tggcgaaccc tgtttctaca aaataaaaat attagctggg agtggtggcg cacacctata 2340gtcccagcta ctcaggaggc tgaggtggaa ggatcgcttg aacccaggag gttgagactg 2400cagtgaactg tgatcattct gctgcactgc accccagcct gggcaacaga gaccttgtct 2460caaaaaaaaa aaaaaaagag acaaattgtg aagagaaagg tactctcata taacatcagg 2520agtataaaat gattcaactt cttagaggaa aatttggcaa taccaaaata ttcaataaac 2580tctttcccct tgacccagaa attccacttg aataaagctg aacaagtacc aaacatgtaa 2640aagaatgttt cttctagtac agtcggtaag aacaaaatag tgtctatcaa tagtggactg 2700gttaaatcag ttatggtatc tccataagac agaatgctat gcaaccttta aaatatatta 2760gatagctcta gacacactaa tattaaaagt gtccaataac atttaaaact atactcatac 2820gttaaaatat aaatgtatat atgtactttt gcatatagta tacatgcata ggccagtgct 2880tgagaagaaa tgtgtacaga aggctgaaag gagagaactt tagtcttctt gtttatggcc 2940tccatagtta gaatatttta taacacaaat attttgatat tataatttta aaataaaaac 3000acagaatagc cagacataca atgcaagcat tcaataccag gtaaggtttt tcactgtaat 3060tgacttaaca gaaaattttc aagctagatg tgcataataa taaaaatctg accttgcctt 3120catgtgattc agccccagtc cattaccctg tttaggactg agaaatgcaa gactctggct 3180agagttcctt cttccatctc ccttcaatgt ttactttgtt ctggtcccta cagagtccca 3240ctataccaca actgatacta agtaattagt aaggccctcc tcttttattt ttaataaaga 3300agattttaga aagcatcagt tatttaataa gttggcctag tttatgttca aatagcaagt 3360actcagaaca gctgctgatg tttgaaatta acacaagaaa aagtaaaaaa cctcatttta 3420agatcttact tacctgtcca taattagtcc atgaggaata aacacccttt ccaaatcctc 3480agcataatga ttaggtatgc aaaataaatc aaggtcataa cctggttcat catcactaat 3540ctgaaaaaga aatatagctg tttcaatgag agcattacag gatacaaaca tttgattgga 3600ttaagatgtt aaaaaataac cttagtctat cagagaaatt taggtgtaag atgatattag 3660taactgttaa ctttgtaggt atgataatga attatgtaag aaaacaacag gccgggcggg 3720ttggttcaca cgtgtaatcc cagcactttg ggaggctgag gcaggcagac tgcctgagct 3780caggagttcg agaccagcct gggcaacacg gtgaaatccc gtctctacta aaaatacaaa 3840aaaattagcc gggtgtggtg acacatgcct gtagtcccag ctacttggga ggctgaggca 3900ggagaatcac ttgaacctgg gaggtgaagg ttgcagtgag ccaagatggc accacttcac 3960tccagcctgg gaaacagagc aagactctgt ctctgagctg agatggcacc acttcactcc 4020agcctgggaa acagagcaag actctgtctc aaaaaaaaca aaacacacaa acaaaaaaac 4080aggctgggcg cggtggctca cgcctgtaat cccagcactt tgggaggccg aggcgggtgg 4140atcacctgag gtcaggagtt ccagaccagc cttgtcaaca tggtgaaacc tccccccgcc 4200gtctctacta aaaatacaaa aattagccag gcgtggtggc aggagcctgt aatcccagct 4260acttgggagg ctgaggcagg agaatcgctt gtacccagaa ggcagaggtt gcactgagct 4320gagatggcac cattgcactc cagcctgggg gacaagagcg agatttcgtc tttaaaaaac 4380aaaaacaaaa caaaaaacca tgtaactata tgtcttagtc atcttagtca agaatgtaga 4440agtaaagtga taagatatgg aatttccttt aggtcacaaa gagaaaaaga aaaattttaa 4500agagctaaga caaacgcagc aaaatcttta tatttaataa tattctaaac atgggtgatg 4560aacatacggg tattcattat actattctct ccacttttga gtatgtttga aaatttagta 4620aaacaagttt taacacactg tagtctaaca agataaaata tcacactgaa caggaaaaac 4680tggcatggtg tggtggctca cacttgtaat cccagtgctt tgggaggctg agacaggaga 4740gttgcttgag gccaggagtt caagaccgac atggggaatg tagcaagacc ccgtccctac 4800aaaaaacttt gtaaaaattt gccaggtatg gtggtgcata cctgtagtcc cagctactcg 4860ggaggcggag gcagaaggaa tcacttgagc ccaggagttt gaggctgcag tgagctacga 4920tcataccaca gcactccagc gtggacaaca gagtaagacc ctatctcaaa aacaaaacaa 4980aacaaaacaa acaaaaaaaa ccacaagaaa aactgctggc tgatgcagcg gctcatgcct 5040gtaatcccag tattttggga ggcccaggtg ggcgtatcac ctgaggtcag gagttagaga 5100ccagcctggc caacatggtg aaaccccatc tctactaaaa atacaaaatt agccaggcat 5160gtggcacgcg cctgtagtcc cagttactgg gaggctgaag caggaggatc acctgagccc 5220gggaggtgga ggttgcagtg agccgagatc acaccactgc actccagcct gggtgacaca 5280gcaataccct acctcaaaat aaaaaagaaa aagaaaagaa aagttgctgt ccccgctacc 5340ccaatcccaa atccaaacag cctctctcat ctcacagtaa gggggaaaaa tcacccaaaa 5400aagctaagtg atcttttgaa aacccaaact cttagaagtc taagattatt atagtcaact 5460catgaagtgt catcataaaa gatactctaa tattatttaa gtagaaccac atattggttg 5520tcttggtatg tctagcccct ggcatacaaa atatttaata acactgatat ggtacctgtg 5580atgtgaaaat gtactatgag tacagcttta taaatactat atatgtacct atatacagaa 5640aaaaatacaa caaaatcata aaagcactta tctttgaaag aggagttaca gcaattttat 5700ttagttcttt attgctttgc tatatattct aaattttttt caatgaatat atatcacttt 5760taaaaaaatt caatggtctt tcttataaat tatctttggc agcatgcgtt tttatatata 5820catataaaat gtatgggaaa tttttaaagg atacattaaa ttaaagcaaa atatacaaac 5880aaaaaatcag aatacaaaaa gataaaaaga ttgggaaggg agggagggag taaggaggaa 5940gggtgggtgg gtatagagaa atataccaaa taatggtaag aagtggggtc ttgacacttt 6000ctacactttt tttaaataaa aaaaattttt ttctctctct tttttttttt tagagacgaa 6060gtctcgctat gttgcccagg ctggtcttga actcctggga tcaagagatc ctcctgcctc 6120agcctcccaa ggtgcttgga ttacaggtgt gagccaccac gcctggtcac tttctacact 6180ttaatatata tattttttca ttttcaatgt catttttatt agttaattta taatacccat 6240tcaccattat attcaaagtc tatttgaaga aataaaccag aaagaatgaa atactctagc 6300tcacatgcta ttcaatacta aattaccttt caaatcacat tcaagaagct gatgatttaa 6360gctttggcgg tttccaataa atattggtca aaccataatt aaatctcaat atatcagtta 6420gtacctattg agcatctcct tttacaacct aagcattgta ttaggtgctt aaatacaagc 6480agcttgactt ttaatacatt taaaaataca tatttaagac ttaaaatctt atttatggaa 6540ttcagttata ttttgaggtt tccagtgctg agaaatttga ggtttgtgct gtctttcagt 6600ccccaaagct cagttctgag ttctcagact ttggtggaac ttcatgtatt gtcaggttgg 6660cccgtaatac ctgtgggaca acttcagccc ctgtgcacat ggccaggagg ctggttgcaa 6720acattttcag gtaggtggac caggacatgc ccctggtcat ggccaggtgg aggcatagtg 6780ctatacagca ggcagaagtc aatattgatt tgtttttaaa gaaacatgta ctactttcat 6840aagcagaaaa aatttctatt cttgggggaa aagattatgc cagatcctct aggattaaat 6900gctgatgcat ctgctaaacc ttcacatatc agaacatatt tactatagaa agaatgaaaa 6960tgggacattt gtgtgtcacc tatgtgaaca ttccaaaaat attttacaac aactaagtat 7020tttataaatt ttatgaactg aaatttagtt caagttctag gaaaatacaa accttgctag 7080atattataaa aatgatacaa tatatattca tttcaggctc atcagaatat atctgttatc 7140acttgacaag aatgaaaatg caccattttg tagtgcttta aaatcaggaa gatccagagt 7200actaaaaatg acttcttcct tgaagcttac tcaccaactt cctcccagtt actcactgct 7260tctgccacaa gcataaacta ggacccagcc agaactccct tgaaatatac acttgcaacg 7320attactgcat ctatcaaaat ggttcagtgc ctggctacag gttctgcaga tcgactaaga 7380atttgaaaag tcttgtttat ttcaaaggaa gcccatgtga attctgccca gagttcatcc 7440cagatatgca gtctaagaat acagacagat cagcagagat gtattctaaa acaggaattc 7500tggcaatata acaaattgat ttccaatcaa aacagattta cataccatac ttatgtcaag 7560aagttgtttt gttttattgc atcctagatt ttattttttt gatttatggt ttactttaag 7620cataaaaaat ttgtcaatac aactcttccc aaaaggcata aacaaaaatt cataaaactt 7680gcatcacttg agatacttca ggtatgaatt cacaactttg ttacaactta ctatatatat 7740gcacacatat atatatattt gggtatattg ggggggttct aatttaagaa atgcataatt 7800ggctatagac agacagttgt cagaacttgg caatgggtac gtgcaggttc attataccaa 7860gtctacttgt agttgttcaa aatgtatcat aatacaaggc cgggcgaggt cgtcacgcct 7920gtaatcccag cattttggga ggctaaggca ggaggattgc ttgaggtcag gagtttgtga 7980ccagcctggg caacagagca agaccctgtc tccaaaaaga aaaaaaataa ttttttacaa 8040aataaaaaca aaatgtatca tcagacgaaa ttaaataaga ggcaattcat ttaaatgaca 8100acttttccca gcttgacatt taacaaaaag tctaagtcct cttaattcat atttaatgat 8160caaatatcaa atactaattt tttttttttt tttttttttg agacggagtc tcgctctgtc 8220gcccaggctg gagtgcagtg gcgcgatcct ggctcactgc aagctccgcc tcccgggttc 8280acgccattct cctgcctcag cctcccgagt agctgggatt acagacatgc gccaccacgc 8340ccggctaatt ttgtattttt agtagagatg gggtttctcc atgttggtca ggctggtctt 8400gaatttccca cctcaggtga tctgcctgcc tcagcctcac aaagcagtag ctgggactac 8460aggcacccac caccacactt ggttaattct tttgtatttt ttttgtaaag acgggatttc 8520accatgttag ccaggatggt ctcgatctcc tgatctcatg atccgcccgc ctcagcctcc 8580caaagtgctg ggattacagg cgtgagccac cccgcccggc catcaaatac taattcttaa 8640atggtaagga cccactattc agaacctgta tccttatcac taatatgcaa atatttattg 8700aatacttact atgtcatgca tactagagag agttagataa atttgataca gctaccctca 8760cagaacttac agtgtaatag atggcatgac atgtacatga gtaactgtga acagtgttaa 8820attgctattt aaaaaaaaag acggctgggc gctgtggctc atgcctgtaa tcccagcact 8880ttgggaggcc aaggcaagtt gatcgctcga ggtcaagagt tcgagaccag cctggccaac 8940gtggtaaaac cccgtctcta ctaaaaatac aaaaaaaaaa ttagccaggc atggtggcac 9000aggcctgtaa tcccagctac tagggaggct gagacatgga gaactgcttg aatccaggag 9060gcagaggtta cagtgagccg agatcatacc actacactcc agcctgagtg acagagcgag 9120actcctgtct aaaaaaaaaa aaaaaaaaaa agatacaggt taagtgttat ggtagttgaa 9180gagagaactc aaactctgtc tcagaagcct cacttgcatg tggaccactg atatgaaata 9240atataaatag gtataattca ataaatagga acttcagttt taatcatccc aaacaccaaa 9300acttcctatc aaacaggtcc aataaactca atctctataa gagctagaca gaaatctact 9360tggtggccta taatcttatt agcccttact tgtcccatct gatattaatt aaccccatct 9420aatatggatt agttaacaat ccagtggctg ctttgacagg aacagttgga gagagttggg 9480gattgcaaca tattcaatta tacaaaaatg cattcagcat ctaccttgat taaggcagtg 9540tgcaacagaa tttgcaggag agtaaaagaa tgattataaa tttacaaccc ttaaagagct 9600atagctgggc gtggtggctc atgcctgtaa atcccagcac tttgggaggc tgaggcgggt 9660ggatcacctg aggccagaag ttcaagacca gcctagccaa catggcgaaa ccctgtctct 9720acaaaaaata caaaaattag ccgggtgtgg tggcacgtgc ctgtagtccc agttacttgg 9780gaggccgagg caggagaatc gcttgaacct aggaggtgga ggctgcagtg agccgagatt 9840gtgccactgc actccacttc agcctgggcg acaagagcaa gactccgtca caaaaaaaaa 9900aaaaaaaaaa aagcttaaaa tctagtggga aaggcatata tacatacaac taactgtata 9960gcataataaa gctcataatc tgtaacaaaa tctaattcga caagcccaga aacttgtgat 10020ttaccaaaaa cagttatata tacacaaaaa gtaaacctag aacccaaagt tacccagcac 10080caatgattct ctccctaagc agtatcaagt ttaaagcagt gattacattc tactgcctag 10140attgtaaact gagtaaagga gaccagcacc tttctgctac tgaactagca cagccgtgta 10200aaccaacaag gcaatggcag tgcccaactt tctgtatgaa tataagttac atctgtttta 10260ttatttgtga cttggtgttg catgtggtta ttatcaacac cttctgaaag aacaactacc 10320tgctcaggct gccataacaa aataccacag actgagtgac ttaacagaaa cttatttctc 10380acagttttgg aggctgggaa gtccaaaatt aaggtacctg caaggtaggt ttcaatctca 10440ggcctcttct ttggcttgaa ggtcttctaa ctgtgtgctc acatgacctc ttctaacaag 10500ctctctggtg tctctttttt tttttttttc ttttttgaga cagagtctca ctctgtcacc 10560caggctggag tacagtggca caatctgggc tcactgcaac ctccaactcc cgggttcaag 10620tgattctcat gcctcaccct cccgagtagc ttggatgaca ggagcccgct accacaccca 10680gctaattttt gtatttttag tagagatggt gtttcactac attggccagg ctggtctcaa 10740actcctgacc tcgtgatcca cccaccttgg cctcccaaag tgctgggatt acaggtgtga 10800gccactgcgc ccgtcctggt gtcttttcat ataagggcac taatccaatc agacctgggc 10860ccaaccctcc cgacttcttc taactgtaat taccttccaa aggccctgtc tccaaatacc 10920atcacactgg gggttaggac ttcaaaaaag gtatgggggg ggtgtgggag gacataaatg 10980ctcagtccat aacaagcacc caacataaaa atggctagaa cagatcacaa aaaaaaggtc 11040ctgtatggct ttggggaagg gctcaacccc aaaatatctg agagctctgg aggggcctag 11100aagtggtaaa tgaatgaaaa cgtggttact ctccagatct gcctttccca aatatggcca 11160ttcttggctg aatcagaaat caaaggacag gttattaatt actagctcta agttacttac 11220catttgctga gacagttcag aaatctgact gcatctcctc agagatctag aacacagttc 11280tcaaattcta acttacttgt gatatacttg tgaatgataa aaatcgctac aggtactttt 11340attaatctga aagagtattg agaaattacc tttcattctg acttttgtct ggaatgaaaa 11400tcaatacttt tgctataatc gattactgaa ataattttac tttccagtaa aactggcatt 11460ataatttttt ttaattttta aaacttcata attttttgcc agactgaccc atgtaaacat 11520acaaattact aataattatg cacgtcacat ctgtaataat ggccttcatg taaacatttt 11580tgtggtttac acataaaatc tctaattaca aagctatatt atctaaaatt acagtaagca 11640agaaaattaa tccaagctaa gacaatactt gcaacatcaa ttcatcatct gtgacaagga 11700ctgcttaagt ctctttgtgg ttaaaaagga aaaaaaaaaa aaagacatgt tggccagatg 11760cggtggctca cacctgtaat cccagcactt tgggaggctg aggtgggcgg atcacccctg 11820gcctgcccaa catggtgaaa ccccgtctct actaaaaaca caaaaattag ctgggcgtgg 11880tggcgggcgc ctgtaattcc agctactcgg gaggctgagg caggagaatt gctagaaccc 11940aggaggcaga gattgcagtg agctgagatt gcaccattgc actacagtct gggcaacaaa 12000agtgaaactc catcttaaaa aaaaaaagac aatgttcgtg ggtccaaaca agacttaatg 12060gaagtgagtc taaaaatgag ctatgtgggc caggcgtagt ggctcccacc tgtaatccca 12120gcactttggg aggccgaagc aggcagatca tgaggtcagg agatggagac catcctggcc 12180aacacggtga aatcctgtct ctacaaaaat tagctgggcg tggtggtgcc tgcctgtaat 12240cccagctact cagaaggctc aggcaggaga atcgcttgaa ccagggagtc ggtggctaga 12300gtgagccgag atttgcatca ctgcactcct gcctggtgac agagcaagac tccatctcaa 12360aaaaaacaaa caaaaataaa agataaaaat gagctatgtg aattaaaaga ggtataacaa 12420tagataaacc atattttatt taattcctag taatgagtaa tatttccaaa cttctggaat 12480gggcagaaat tgctagttgg catattttta ccttttatat tcagatacat taaaattctc 12540aaaaaaaaac acctcaaagc agatgatccg ccatctcctt ggataatttg tgttaactca 12600ggataacaga aaaccaaaat tatgagttac tgatgcaata ttcctaaatg taaaaataat 12660taaagctaat agtagattca tcttccaatt tcatatcagt cttacaaata aactacatat 12720ataacttgct tgccttccct tctgagggat aaagctgtta gaagaattaa aatcagcatt 12780cttgactatt caaccaaggg agggataaat tattactcat tctagggaca tgggctcata 12840actactacat gtgtaaggac atgaatttac ccaatattac aatttttcct tttattagtg 12900tgtacagtgg aagaatagac atgttcactc tggacaaaaa aaaaattata cttatcagtt 12960atcagaagca caatgctgaa gacagtagtt ccataacaat ttgaagtatg tgatcgaact 13020agtagattat cttagtagta gtgaattatt gtaaatgtta gtaatttggc agccactggg 13080cagaaaaata agaattgagg ctcaatattg atattaatgg tggtgattga cacataaatt 13140ttatcaagtc tacacaatat aaaattacag aaaggtagaa gagtatacca gtacaacttc 13200aacatatctt cactacaagg gagtaaaatg acatggccta gttactatct aatgaactgc 13260agaaaactaa aagaaaactc caaggcaact cttctctgct gatctggttg gtccttttcc 13320taccttttgc aatacccaga tacaaacaat ggatagaaaa caaagtagac ttgtagtatg 13380caggtcacag tgctaaattc acagaaagaa acccctgaac tgaactgctc tatttcctgg 13440tggtcacaaa gagtaattct ggtttacacc tacagattga tgtcaatcta caccctgttg 13500ataacagtgt ggccaaggac aaaaaaaagg tgctccgttt taccaattct gtaaaaaatt 13560attggcaggg taagctcggc tagggcagga ttacatttct aggactacca tccccgaaat 13620ttagaagata ttatatccac ataaagcata tctttcacat taatttgcaa aaatctaaaa 13680gctttttctt agctcaagtg tgtccaagtt taccctggca gtttaaaacg atagttacaa 13740gcagcatggg ttgtatcaga cacatttgag ggccaatttc atgtaagtga tattgggcaa 13800gttacttcaa ctatctgtgc ctccaaggtc atactagtgt ttatttacct aaagggtacc 13860tgttatgtaa ctttagggtg tttacattag ataatgcctg caaaatattt acttcaacgc 13920ctaaaacata gttaagtatt caataaatac ctactattgt cactactaac ttaaaagttt 13980agagattaag agcagaatct ggggtgagac aaacttaggt tcaaatccta gtattgttgg 14040gtaatcttgg gcaagttact taacctctct gatttgtgta atttaaaaaa ttagttaata 14100tacataacag ggcttagaag agtatctagc acatagcacc atttaagcat ttgttattgc 14160taacatgcaa acaatttaag ggaaagaaat tttttaaaaa ggaagaggga tttgcaaact 14220aaaaacaatg agtatcttat gttcaaagaa aactaacaaa cagccagctc tagcaataat 14280taaattcact atatactggg gcaggcatca caccccaaag ctaaaagcgt ctacctaggc 14340caggcacggt ggctcatgcc tgtaatccca gcactttggg aagcagaggc gggcagatcg 14400cttgagctca ggagttcaag accagcctgg acaacatggc aaaacaccat ctctacaaaa 14460aatacaaata ttaggccggg cgcagtggct cacgcctgta atcccagcac tttgggaggc 14520caaggcgggt ggatcacctg agatcaggag ttcgagagta gcctggccaa catggtgaaa 14580cctcgtctct attaaaaata caaaaaatta gccaggcatg gtggcaggcg cctgtaatcc 14640cagctactca gggggatgag gtaggagaat cgcttgaacc cgggaggcag aggttgcact 14700gagccgagat catgccactg tactccagcc cgggcaacaa gagcgaaact ccatctcaaa 14760aaataaataa ataaataaat aaaataaagt

acaaatatta gccagggatg gtggtgcgca 14820cctgtagtcc cagctacttg ggaggctgaa gtgggagaat cccctgagcc tggggagaat 14880cacccgagcc cgggaagtcg aggctgcagt gagcagtgat tgtgccactg cactccatcc 14940taggtgacag agtgagaccc tgtctcaaaa aaaagaaatt ggcagaatta agtaagttga 15000tgtttagaga tgaaaaatca acattttttc ctcagcaact gaataaaaac aacagccact 15060accatttttt tgagtaccta tttgtagcct attttttaac tggtattact cgagagagag 15120agagctaggt tcgagacaga gctccttctc ttaataactg tatgacctag ggtatgtctg 15180ttagcctctc tgaggcttca aaggttcctc atctgtaaaa tggtaataat cataccattg 15240ctacagggct gttttgaaga ctaattagga ctatgtaagt aaacatgatg atggctatta 15300ttactgttcc ccgccagggg ccatgcaagg gttgctgatt cacatagact gtcttataat 15360cctctcaata actccaagag gtagccagca cctcagatat acataaaatg acttaagccc 15420agagaggtga agtaagttgc ccacagccac acaactagta aatagcccaa acaagctgga 15480ttcccagtta gactccgtta atagcactgc tctttacctt aagtcattac aatgcctaat 15540atgaaataga atcgcttctt tcttagggtt caagtggtta attatttaat gtattcattc 15600aacaaaccat catcgaggac ctcttacaag ccaagtactg tgctaagtgc tagagttacg 15660gcggtgattc ctgcccttaa aaagttttag tgggagaaac aacaggtaac caggtcattg 15720ccaaaacaac aaaaataatc ataataaagc aggctaaagc atatttaact ggccggggtt 15780ttgactattt tagcaagcat gatcagaacg gttgaggagg gaggccagca gcttggccgg 15840ttcaacaaac aagaaaaaac cagtgagggt ggagctaaga taccagaggc tgattacggt 15900taagaatgtt cttgaaggta aggaccagat tctcattttc tatatcctgg ggcatcggtc 15960agcatggaat ctggattcta gcacatgtga atttcggctt gaaatgacct aatgcctttt 16020ccctagttcc ttcgtgtgtc aaatacgcat ggttaccgct accagagctg tagtggggct 16080tcaatgaggc catgagcatc tccataaaga tgaactacag tgtgtgcaaa actaaaggca 16140aaacctggtc cccacacgcc ctcccaggtg gtcgctttcc gtgccgaggc ccctccagag 16200gtgccccgag aacctcacca tcgcacccca aacttccagg gaagggcctc tcccgagaaa 16260gcccccacgc ccccaccccg cgccatcatt cccgaatctg ccctcggccc ctccccgcag 16320cacgctcgca ggcggcacat gtcaaccaaa acgccatttc caccttctct tcccacacgc 16380agtcctcttt tcccagggct cccccgagga gggacccacc ccaaaccccg ccattccgtc 16440ctccctgccg ccctcgcgtg acgtaaagcc gaacccggga aactggccgc ccccgcctgc 16500ggggttccct gggcccggcc gctctagaac tagtggatcc caattgaagg cctggtctaa 16560atgactccaa aatcaccact taattcaaga gactgatttc cctgagtcag gccccttaaa 16620gcagctattt caatgggaca gggaaacaac cctaggatct ggattagaat cacttggggg 16680ctgccacacc cccagggctc tgatcctgcc cttctcccac acgcacattc acatactgct 16740gcagtgacct tccatttcta atgggttcct gggccatctg tcaggtatag ggaatggaaa 16800aggggttggg gaggctctgc ttcagaaagt ttgtgtcagg ggctcccaga gcctccacag 16860atagatagca ggggtcccca ccctaccatg gcagctataa atgtgatcaa catttattgg 16920cctaggatac agcagttagc aaaatgcctg atgtagttcc cactccgtgg aggttgcagg 16980ctagccaaga agtcatgagt tcagcaaccc ttacgcacca gtgggatgag attggaccag 17040gccgagggta gtcttgggaa cactcagcat ttgtctgagg gccagaagag gctgcttgcc 17100ctcagacagg aggtcagcat ctttattgta gcccatgaca cctctacacc attgctcttc 17160tggtcttatg gaagacatct ttgggcctga taacagcgga gtctgtgtcc cacttgtcca 17220ggctggagtg ccacatcagg cacactccag ttgcagggac agcacagaca agtttcagga 17280aggctggtgg cctccaggag gttaacctta taaggccaga ttgtaaccta gttgaaaaac 17340atacacatgc catgataata aaagaaccta ggcaccatta caagagaaaa aatcattttt 17400gtagatacga gcatggattc ttgggtgggt cagacacact gggcttgtgc tctgactgca 17460ctgtctcccc tacctgacct tgggtaaacc ataagactgc tgcatgactc agtgtccacc 17520ccaaaaaagt accggtagat attggccaca gtagatatca gctagagtgg actctcatga 17580caatgagggg agatgtattc cccatcttag gcacctggga ctctaccttc catcttctgc 17640tccgtgtctc tccatcccca ggctcttcag aactcaggga gtccagaatg tcagctccca 17700gatttcagcc ttcagaaagg aaacccatta ccgttcagtt gaacaaatgt tgtctgagcc 17760ccagatctgg gctcagaggc catctaggct atgagacaag aggggaacaa agcaccgtct 17820gcactcactc accacactca cttgctgtcc caggtcacat ccatcgggta gagaatctaa 17880gaggctgagc tagctcccgc caccagccca gcccacccca cctggcccct tccttccttc 17940tacaaaatat gcaccacctg tcaaagggtg ggcagtgcca ggcctgcata cagagcactg 18000agtgtaaaag cagacatgga ccctgacctc caggagcttc caattttctt gaagagacaa 18060atcagctggc atttcagtcc agtgtgatct gctcttggtg agcacagacc tagggagttg 18120gggcagcttc ccagaagaac tgcagtccag gctgagggca gagaaatgag gggaatggcg 18180aggaattggg gagcaggggg gagctcagta gagagccaag ggcgggaggt gagaagtccg 18240tgttgggcca ggagctaccc tccggtggcc acagccgaag tcgaggatgc ctttggaact 18300catccccact tctctctttc tgtatgtagc cgtccaagaa caagtcacct ccaagtgtag 18360ccggatcaag gcaagccccc catctagcaa gcacttgatg ccacccagaa ctgggcttct 18420tcagaacaat ctgagtccag gaatgatccc actcaccagg caccagagct gcgagggcat 18480gggagtgatc tcaccaactc tggggaagcg gcaaggaatt ttcacctcca gcccccagtg 18540tcccatcctc tcacactcag gccagactcc cctgggcaga cttgactctg tctgccagca 18600tatgcagagc cccaaggcca ccccaccaga agtgcccctg cctgggttct gtcccagctc 18660cctgggcacc cagtccttga gtccccacca gctcagacgg cctagtgtgc caagaatgcc 18720cactgcgttc aacaatgctg catgggtcac agcggcagca gctgtgacca cagcagtttc 18780ggggaaaaca cccctcagcc aagtggataa tagcgttcag cagcactcac cttctggcca 18840ggcctgcctt cagaggccat ctgattggga ggcacaagtg cccgctgcga tgggaacaca 18900agtgcccctg gccaacaacc ccagcttcag cctgctgggc agccagagcc tcaggcagag 18960cccggtacag ggcccggtgc ctgtagcaaa caccaccaag ttcctccagc agggtatggc 19020cagctttagt cccctgagcc ccatacaggg catcgagcca ccaagctatg tggctgctgc 19080tgccaccgct gctgctgctt ctgccgttgc tgccagccag ttcccaggtc cgttcgacag 19140aacggatatt ccccctgagc tgccacctgc cgactttttg cgccagcccc aacccccact 19200aaatgatctg atttcgtcac ctgactgcaa tgaggtagat ttcattgaag ctctcttgaa 19260aggctcctgt gtgagcccag atgaagactg ggtgtgcaac ttgaggctga tcgacgacat 19320tttggaacag catgctgctg ctcaaaatgc cacagcccag aattctgggc aagtcaccca 19380ggatgctggg gcactttaaa tctgagcagg atgcccatag aaacccccat ggtgacatca 19440ctctaggaag tggtgtcgat ccatacccgc agttgtctcc cgttacaatt tgagtggtgt 19500tgtcagccca tgcttatccc tctctctacc tgtgacaaaa tggaaagctg gtgatttttc 19560aagctacgtg tacatatttg aaaattttgt aaatggtttt cctaaacatt aatgacagaa 19620gtatttatac ttcattttgt gactttgtaa ataaagcgac ggcttttgtt tcagtagagt 19680tgtgtttact atgcattgtt ttgtgtttat tatacaatgt tacaaatatg cagaccgtgt 19740tgtttgctcc agtgatacct tgttaagcta ggtggctgag tcgcttatgg ttttaatgca 19800atgagcaatg tggatatgac caagagttgt tgtgcaagtt gacaaatgcc aaatagaaaa 19860ccacttggcc atttatttct atgttcacta aaaatcctat tgccttgtgt gattcttaat 19920ctcttttgcg aacctttcag tctccgctag ctctttccta atgagcttta cagcagaagc 19980tgttttatcg ttaagtgccc cacagagaca ctttaccagg aggctgggag agttctccag 20040atttgggaga ggcgcagaga cagtgtgtga gccgagccct gtctcagcaa tccacctgga 20100ggagctagag tatcctcctc cctttaccat tcagaccgag agaaaaagcc cagcttgtgt 20160gcaccctcgt ggggttaagg cgagctgttc ctggtttaaa gcctttcagt atttgttttg 20220atgtaaggct ctgtggtttg ggggggaaca tctgtaaaca ttattagttg atttggggtt 20280tgtctttgat ggtttctatc tgcaattatc gtcatgtata tttaagtgtc tgttatagaa 20340aacccacacc cactgtcctg taaacttttc tcagtgtcca gactttctgt aatcacattt 20400taattgccac ctcgtatttc acctctacat ttgaaatctg gcgtctgttt caagccagtg 20460tgttttttct tcgttctgta ataaacagcc aggagaaaag tgcctctatg tttttatttt 20520tcaagggagt attcagtacc tacaaaccca agtcaggaag cctgctagtg gctttggttc 20580tttcagaggc tgctcgatgc cttgtgtgtc agaaagaaag attcagcagt tttgcatcat 20640ggcaaagaag cctgttattt tggggctcag cccctcattt tatagaggat gaaacagagg 20700gggatgggag gtcacaaaga caactgcccc gggagcaggt gtgggggaga cttgccctga 20760gggtctagac gctctgcacc accgtcctgt ctcccttgct gaagaccaca catgcccttc 20820tttgaccaga ccctgccacc tgataggcca ggacctggta ggcgggtacc caggtttcat 20880ggatggaacc acatctcccc aaaagtgggg aggtagctac tgggatgcac gcctcccgcc 20940atgtgctata ggagagcagc tgaagcaaca gttgggatca gatgtagtca caattgaatg 21000catcatcaca tttatccctc taagtggctg ggagagttga tatcctcatc cctaaggtac 21060aaaatgttcc aatttgatca gtggctttca ggagctgaga aaggcatgtg ctctgaggca 21120gagctgttat gtcccgcaga gcctaaaaat gctctaagaa catgctccct gccaaaattc 21180tcaatggctg tgacaaggga caacgatcga ccaatggggg tggaagcaga cctccgcagt 21240ccaggggcca gagctaggac agaggggtcg gagaaagagt cattttccca acactccagc 21300tcttggccag tcctcacaca gtcccctcct gcttcctgct gagagagata tcctcatagg 21360tctgggtaaa gtccttcagt cagctttcat tccctgtcac caactttgtc tctgttctcc 21420ctgcccgtct caggcagcac tcctcaggaa acctctccaa gagccagcct cactgcagcg 21480cccactattg tccctctgcc tcaagtgtcc catccatgcc aggccccagg caggctgcag 21540ctttccctca gggccacacc aaagcacttg ggctcagctg tgctgtcccc ctccatcact 21600gagctcaggg gcagcagggg tggggtgcca ggaggcccat tcacccttct ctggctctgt 21660gttggaccca cctgcccagc cactgctgct tagaacctac ccgctgggaa aatgaagccc 21720tcccggaggg gccacctcaa cctgagagcc tcacggatca cagttgtccc cactcagctc 21780tgccagccct cagagaccca tagataaaag ctgagcttgg ctcgcagagc tggttccatc 21840ttccattccc agagggttca acttcctacc ccaaccacac agggaacctc aaggctgagc 21900cagtgtgggc tgcagtgcag accagcttcc tggacacgtc ctgccacctg accccaggct 21960ggcctcactg cccctggcac tcctgaccct atcctcattc ctcctggcag tgcgtgttct 22020gccattccgc tttcccttag ctgtcctctc actgtactgt cagcttctcc ttttccaggt 22080gccccccagg ggctttccac atgaccctgt caccccacag cccatccagc accaattcca 22140gctctctgcc acccttcaaa ggagtgacag tgccctgctt cacctcccac tcacccctca 22200acccagagca atctggctcc agtcttgcct ccttccccct aagtactcta gtcacagttc 22260caaattcctc ctggtcataa agccaaatga agcttcctgg tcctcagcgg acttgccact 22320tcagcagtac tggactctct cctcccagaa acctgtttcc ccttggctcc tggagcccac 22380actctgctgg aatccttctg cctctctggc ctgtagcctg gccctctctc ccaacctgag 22440gtccattctc tcctgctcct ccacaagatg ttgctccttc cattacttcc tccctctcaa 22500ccaaagctcc ttcattagct ctttatcttc tggtttcttc ccctgggcag acgaatggat 22560tcaagagcct gtggcccagc agcccagcac tccaggatct cagcacttca gcatcccagt 22620accctagcat ctcaataccc cagcacccca gcaccatagt attccagcac cccattgtcc 22680aagcatctca gcactccagc atcccagcac cccaacactc cagcagccca gaatctcagc 22740accctagcac tgcagcatct caggacccca gcacttcagc atcccagcac actagtactc 22800cagcatctcg gcaccccagc acctaggcat cccaacaccc agcaccccag cacttaagca 22860tcccaccact acagtatctc aacactccag caccccagca ccatagtgtt ccagcacccc 22920agcatcccaa caccccagca cttaagcatc ccaacacctc ggcatcccaa caccccagca 22980ctgcagcatc tcagcacctt agcatcccag tgccctagca tctcaatgct ccagcacacc 23040agtactacag tattccagca ccccagcact ccagcatctc agcactgcag cactgcagca 23100ctccagcatc ccaaaatccc agcatcccaa caccccagca gaccagcaga ccagcatctc 23160agcaccgcag catccaagga ctatcccagc atcccagcaa cccagcacct cagcatccca 23220acaccccagc atttcagcat ggcaacaccc cagtacccca gcacttcagc accccagtat 23280cccagcatct cagcgaccca gtatcacaaa acctcagcat cctagcaccc cagcacccca 23340gcaccttagc accttagcat cccagcatct cagcgcctca gcatcttgat attctggctg 23400aggtcagcgt ggtgtatcta gtcagggtcc taactttcac ttcgcaggga aatgctgctg 23460gactgggtct catgttgggc tgaagctctc tagacccctt gaagacagca taaaagagct 23520tggagacgct gggtgtcccc catggaagag ttcactctca tcctgctttg acaacagcct 23580tctctggggt ccctcacggg cccctctttc ttactgcaag tttgtctctg agaagactgt 23640gatgcagaag tcactcagct gcctgtggct cctgaagagc tgaaggtgga ggcctgtagg 23700cctccctatg agaggcgcag aaaaaaccat gattgctagt ggggaggtgc tccctctaca 23760acccactcca taatctgccc ccgcccagct ctgaggccag ccccagggga aaatgccaga 23820tccccaggga ggtgtgtgag acctcagggg ctccctcctc ccttacagca ggctcaggcc 23880cctgggggcc tcagggccaa ggtctgtggg taagctacta tctctcactt gtcctctagc 23940cacaaaagcc agggagatct ggcaatggac atgaggttct gaagaagcac atatgactgg 24000cttcctaatg cgtggttgtt cagtgattca ataaacacgc atgggccagg catggggaaa 24060tagacaaaca tgatccccaa cctctcccag agtgaactgg gagggaggag tgttcatccc 24120tcaggattac accagagaaa caaaccagca ggagatatat atggttttgg ggggtcaaga 24180aagaggaaaa acctggcaag gcaagtccaa aatcatagga caggctgtca ggaagggcag 24240cctggaacct ctcaagcagg agctgatgct gcagtccaca ggcagaattt cttcttcctc 24300ggggaaatct cagctttgtt cttaaggcct ttcaactgat tggctgaggt ctgccccttc 24360ccccacattc tccaggataa tcttccttac ttaaagtcaa ctattaatca cagctacaaa 24420atcccttcac agctacacat agatcagtgt ttgattgacg aacagcccct acagcctagc 24480caagttgaca cataaaacta accatcacag ggggacaaat gatgtaaaca catcaacaaa 24540taaaacagta acaagttaag gtctatggaa aaaacacaga aggggcagag agaaagaaag 24600caagaaggag agtcccagtt tgctagggct tgtgggaagt ggggagcagt tctctttagc 24660taggatattt gggaaaggca tatctgaagg agtgatattt gagcttagat taaaagatgg 24720gaaggagcaa gccatgcaaa gagctaggat gttccaagca gagacggaac agcaagtgca 24780aatgtcagga ggaatagaag gaggctggtg ggtggggtcc agtgagcaag aggagggcag 24840gcaggagagg ggatggggag gtgggcaggc ccagaccacc cagggccctg gagactatcc 24900tgatccaaca agggaagcct tgagtcactt cagtgtccat gtggagaatg gacctcagac 24960tgaatgaggg aggcagtaag gagggcctct acctccaggg cttcgccctg tggactgcgc 25020atagacatct ccaactcaga aagtctgaac caaactttcc atagttcccc caagtctggg 25080catcctccta ctcagtgaaa ggcagccatc acacctccct gccctgctcc cggatgcccc 25140aaatcctctt ggtctccaag tccagaacct gagacttgtc cttgatgttt gtctttccct 25200caccctttct gtattctggg aagatgggtt tttttccccc agatgaatct gtaaaacttc 25260tgtgatcaca ataaaaattc tggcagtatt attttctgga acatgacaaa gtgattcaaa 25320attatttatc tggaagacta caaaacaaga atagccagga aatttctaaa aagaaagaag 25380aaggaggagg agaaagaagg aggaggaaaa ggaggagaag aagaaaagaa aaagaaccaa 25440gaaagggttc tagctctacc aaatattaaa acatatcatg aagctattta aaacaatatg 25500gttgtggata ctgaaaaaga tgtgaataaa gtggaaggaa aataaataga aatgcacatg 25560gggattgaga ctgtgaaaaa ggcagcatct cacatcagtg agggatgttc aacacctggt 25620gttgggaaaa ctggctagtc atttaaacca aacaactggg tcctctacct cactcctgac 25680attaagatac atttagatga ttcaaagagt aagacagaaa aaataacacg tgaaaacact 25740atcagaaaac aacgtgggcc aggtgtggtg ggtcacgcct gtaatcccag cactttggga 25800ggccgaggca gacagatcac ctgaggtggg gagttcaaga ccagcctgac caacatggtg 25860aaatcctgtc tctactaaaa atacaaaatt agctgagcgt ggtggcgcat gcctgtaatc 25920ccagctactc aggaggccga ggcaggagaa tcacttgaac ctgggaggca gaggttgtgg 25980tgagccgaga tcacgccatt gcactccagc ctgggcaaca agagtgaaaa tccatctaaa 26040aaaaaaaaaa aaagccaagg tggatatttt tatagtatca gggtagatca agcttctcca 26100atcatgacat gaaacccaga aaccataaaa gaaaagaatg ataaaattgc ccacgtaaag 26160taaaaagctt gcacacagaa aaacaccata caggttacaa gatgagcagc aaaatcagag 26220aaaaaacatt gcaattcagg acacacagag gctattgttc ctaatattta aaaataaaag 26280tagtggattg tctacaaaaa gatgaagaca agaatttcag aaaaccaaat actgcatgtt 26340ttcacttaca agtggaagct aaacactgag tacacgtgta cacaaagaat ggaaccatag 26400gccaggcacc gtggctcacg cctgtaatcc cagtactttg cgaggccgaa gcgggcggat 26460cacctgaggt gaggagttcg agaccatcct ggccaacatg gtgaaaccca gtctctacta 26520aaaatacaaa aattagccgg gcgtggtggt gggtgcctgt aatcccagct actcgggagg 26580ctgcggcagt agaatcgctt gaaccctgga ggtggacctt gcagtgagcc gagatcgcac 26640cactgcactc cagcctgggc aacagagtga gactccatct caaaaaaaaa aaaaaggaat 26700agaacaatag acactggggc ctacttgagg gaggagggtg aggatcaaaa acctgcctat 26760caggtactat gcttattacc tgggtggtga aataatctgt acaccaaacc ccagtgacat 26820gcaatttacc gatgtaacaa acctgcccat gtacccgctg aacctaaaat aaaagttgga 26880aaaaaatata gaaattttct ttgtaatagc caaaaactgc aaacagccca ggtgtctatt 26940agtagaatgc ataaacaaac tcgggcatgt tcatacaatg taaaactact catcaataaa 27000aagtgatact tctcagcaat gaaaagaaac tagctactga taccagctac aacatggatg 27060gatttcaagt gctttatgat gagagcaaga agccagacac aaaagtgtct atatatatat 27120acagtatata tacgtatata tacacatata tacagtatat atatacatat acatgtatat 27180atatactgta tatatactgt atatatatac acagtatata tatacatata tacagtgtat 27240atatactgtg tatatataca tgtatatata ctgtgtatat atacatgtat atatactgtg 27300tatatataca tgtatatata ctgtgtatat atacatgtat atatatgtat actgtatata 27360tactgtatat atatatacac atatatacag tatatatata cagtatatac tgtatatata 27420cagtatatac gtgtatatat acatatatac agtatatatg taaatataca tatatacagt 27480atatatgtaa atatacatat atacatgtat atatatacac tatatatata catatatagt 27540gtatatatac atatatacat gtatatattt actatatgat tccatttata taaagtgcca 27600aaacagtcaa aaataatcta tgtggaaaaa atcaacaaag ggatcccccg ggctgcagga 27660attcgatggc gcgccgacgt cgcatgctcc tctagactcg aggaattcgg taccccgggt 27720tcgaaatcga taagcttgga tccggagagc tcgataacat ttaaatggat ctaccacatt 27780tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa 27840aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag 27900caatagcatc acaaatttca caaataaagc aatagcatca caaatttcac aaataaagca 27960tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 28020gagctaggat cccgcggccg cctactggtc ctcctggagg tagaacttgg tgaccatgaa 28080ggactctttg ggcttgttgg tgaggctgac gggccggtct gcctcctgcg ccgtgcagag 28140gaaccagcca gggcaggcgg cagactcgaa gctggtggtg gggccactgt ttgagcggat 28200gaaggtgaag cgcttgttct cctccttgtt cttgctcagg tcagtgatgt taactgcctc 28260caattggaac ctaatctcat caccagactt gacacaggcc aggcacagct tcctcccatg 28320gagtcccagg aatagagcat caggctcaat gggcaccaca tctatcttct cttgtaattt 28380agtatttgat tcttgcaagt atccagcaac tagttggtta ttcctcatgt agaaggtctt 28440ctggttaaca tcccagattc tgaaggcttg catcttgcag ggtctcttcc ccaaagggtg 28500gcaggctgtc tctgagtaga gcaagaaaag gaggagagag attaggtgtc tgacagaacg 28560cctgcggatt tccataccgg tggatccatg gctctgtctc aggtcagtat aggagctttg 28620atgtgaagtc agccaagaac agctgaacac tacttcctct gaggcccttt tataggaggg 28680attgcttcct gtgaataata ggaggatatt gtccacatcc agtaaagagg aaatccccaa 28740ctgcatccaa aaagttttct gggaatatcc actgctgcag gagtggaaag tccccagtgg 28800aaagtcccca gtggaaagtc cccagtggaa agtccccagt ggaaagtccc cagaatttcg 28860acggcgcgcc taccagtaaa aaagaaaacc tattaaaaaa acaccactcg acacggcacc 28920agctcaatca gtcacagtgt aaaaaagggc caagtgcaga gcgagtatat ataggactaa 28980aaaatgacgt aacggttaaa gtccacaaaa aacacccaga aaaccgcacg cgaacctacg 29040cccagaaacg aaagccaaaa aacccacaac ttcctcaaat cgtcacttcc gttttcccac 29100gttacgtcac ttcccatttt aagaaaacta caattcccaa cacatacaag ttactccgcc 29160ctaaaaccta cgtcacccgc cccgttccca cgccccgcgc cacgtcacaa actccacccc 29220ctcattatca tattggcttc aatccaaaat aaggtatatt attgatgatg ttt 29273

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-.kappa.B 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-1.alpha. (MIP-1.alpha.) 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-.kappa.B 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-1.alpha. (MIP-1.alpha.) 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.