Methods for treatment of inflammatory joint disease

Abstract

To provide compositions and methods for preventing inflammatory diseases of joints, including rheumatoid and osteoarthritis, tendonitis, bursitis, inflammation of a ligament, synovitis, gout, and systemic lupus erythematosus.SOLUTION: A therapeutic anti-inflammatory composition for treating an inflammatory joint disease comprises a therapeutically effective amount of an IL-10 expression construct, and is administered by injection into an inflamed joint. The IL-10 expression construct comprises a bacterial or viral backbone and a nucleic acid sequence encoding interleukin-10.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to treating clinical conditions associated with inflammatory diseases of the joints and associated symptoms thereof by administering to the inflamed joints therapeutic anti-inflammatory compositions comprising bacterial or viral interleukin-10 (IL-10) expression constructs. [Background technology]

[0002] Statement of Federally Funded Research This invention was made with Government support under Grant No. U44-NS071642 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] In the following description, certain articles and methods are described for purposes of background and introduction. Nothing contained herein should be construed as an "admission" of prior art. Applicants expressly reserve the right to demonstrate, where appropriate, that the articles and methods cited herein do not constitute prior art under any applicable provision of law.

[0004] Joint pain can break out for many reasons, for example as a result of a wide variety of joint conditions (of which rheumatoid arthritis and osteoarthritis are the most common), as well as tendonitis, bursitis, ligament inflammation, synovitis, gout, and systemic lupus erythematosus. When injured, a chain of events in the immune system known as the inflammatory cascade is triggered, causing redness, swelling, and pain. Then, once the threat is reduced, anti-inflammatory compounds take over to heal the affected area. When this process (known as local or acute inflammation) occurs, it is a sign of a healthy immune system. However, prolonged inflammation can lead to a more chronic condition. Summary of the Invention [Problem to be solved by the invention]

[0005] Current treatments for rheumatoid arthritis and inflammatory diseases of the joints, such as osteoarthritis, tendonitis, and synovitis, remain suboptimal. Finding treatments that require less frequent administration could significantly impact the quality of life of patients with inflammatory joint diseases; however, despite substantial research and development of treatments for these conditions, there remains a large unmet need for treatments that are safe, effective, and easy to administer. [Means for solving the problem]

[0006] The present invention provides methods of treating inflammatory diseases of the joints, symptoms associated with inflammatory diseases of the joints, and slowing disease progression by administering to a subject, typically by injection into a joint, a vector expressing an interleukin-10 (IL-10) coding sequence. In some embodiments, the IL-10 expression construct is encapsulated in biodegradable microparticles, and in some aspects of these embodiments, the microparticles are suspended in a diluent to form a therapeutic composition.

[0007] In yet another alternative embodiment, the therapeutic composition of the invention is an IL-10 expression construct (with either a bacterial or viral backbone) that is delivered as "naked" DNA, i.e., the IL-10 expression construct is delivered without encapsulation. In such embodiments, the IL-10 expression construct is delivered together with one or more diluents, and in preferred aspects of these embodiments, the IL-10 expression construct is delivered together with one or more adjuvants. In some aspects of this embodiment, the one or more adjuvants may be administered to the subject simultaneously with administration of the IL-10 expression construct or up to 10 days prior to administration of the IL-10 expression construct, e.g., as a "pre-treatment."

[0008] In some aspects, the arthritis is in the knee, elbow, wrist, ankle, hip, shoulder, or spine. Conditions treatable by the methods of the invention include rheumatoid arthritis and osteoarthritis, as well as tendonitis, bursitis, inflammation of the ligaments, synovitis, gout, and systemic lupus erythematosus.

[0009] Accordingly, some embodiments of the present invention provide a method of treating an inflammatory disease of the joint or a disorder or condition associated therewith in a subject, the method comprising administering to the subject a therapeutic anti-inflammatory IL-10 compound comprising a bacterial backbone and plasmid DNA comprising at least one IL-10 coding sequence, and a diluent, wherein the therapeutic anti-inflammatory IL-10 compound is from about 1 μg DNA to about 1000 μg DNA, or from about 5 μg DNA to about A therapeutically effective dose of 900 μg DNA, or about 10 μg DNA to about 850 μg DNA, or about 20 μg DNA to about 800 μg DNA, or about 25 μg DNA to about 750 μg DNA, or about 40 μg DNA to about 500 μg DNA, or about 50 μg DNA to about 250 μg DNA, or about 5 μg DNA to about 200 μg DNA, preferably about 2.5 μg DNA to about 500 μg DNA, is delivered to the joint. In another embodiment, a viral vector is used instead of a bacterial vector.

[0010] In some embodiments of the present invention, the IL-10 therapeutic compound of the present invention (i.e., encapsulated or naked DNA) is administered together with one or more adjuvants. The adjuvants of the present invention are biocompatible agents that can be administered simultaneously with the IL-10 therapeutic compound or as a pretreatment prior to administration of the IL-10 therapeutic compound. Particularly preferred adjuvants include, but are not limited to, mannose, sucrose, glucose, calcium phosphate, dendrimers, liposomes (such as cationic liposomes), and oligodeoxynucleotides. In a preferred embodiment, the adjuvant of the present invention is an adjuvant that increases the uptake or efficacy of the IL-10 expression construct. The co-administration or pretreatment comprises administering to the joint about 5 μg to about 1000 μg of adjuvant, or about 10 μg to about 750 μg of adjuvant, or about 50 μg to about 500 μg of adjuvant, or about 25 μg to about 750 μg of adjuvant, simultaneously with or up to 10 days prior to administration of a therapeutic anti-inflammatory IL-10 compound.

[0011] Other alternative embodiments provide a method of treating an inflammatory joint disease comprising administering to a subject a therapeutic anti-inflammatory composition comprising an IL-10 expression construct; microparticles encapsulating the IL-10 expression construct; and a diluent, wherein the therapeutic microparticle compound delivers a therapeutically effective dose to the joint of about 20 μg DNA to about 1000 μg DNA, or about 25 μg DNA to about 750 μg DNA, or about 50 μg DNA to about 500 μg DNA, or about 50 μg DNA to about 250 μg DNA, or about 50 μg DNA to about 200 μg DNA, or about 25 μg DNA to about 100 μg DNA.

[0012] Yet another embodiment of the present invention provides a method of slowing the progression of an inflammatory joint disease in a subject, the method comprising administering to the subject a therapeutic anti-inflammatory IL-10 compound comprising plasmid DNA comprising a bacterial backbone and at least one IL-10 coding sequence, wherein the therapeutic IL-10 expression construct is about 1 μg DNA to about 1000 μg DNA, about 5 μg DNA to about 900 μg DNA, about 10 μg DNA to about 850 μg DNA, about 20 μg DNA to about 800 μg DNA, or about 25 μg DNA to about 750 μg DNA, or about 50 μg DNA to about 500 μg DNA, or about 50 μg DNA to about 250 μg DNA, or about 50 μg DNA to about 200 μg DNA, or about 25 μg DNA to about 100 μg DNA, providing a therapeutically effective dose to the joint. In such embodiments, the adjuvant is preferably administered together with the therapeutic anti-inflammatory IL-10 compound or up to 10 days prior to administration of the therapeutic anti-inflammatory IL-10 compound, eg, as a pretreatment.

[0013] Optionally, in the described embodiments, the IL-10 expression construct comprises at least one nuclear targeting sequence located either 100-2000 bp 5' of at least one IL-10 coding sequence and / or 150-450 bp 3' of at least one IL-10 coding sequence. In other aspects of the invention, the IL-10 expression construct comprises two nuclear targeting sequences, one nuclear targeting sequence located 100-2000 bp 5' of at least one IL-10 coding sequence and one nuclear targeting sequence located 150-450 bp 3' of at least one coding sequence.

[0014] Preferably, the therapeutic composition is administered by injection into the joint, and in a preferred embodiment, the therapeutic composition is administered by intra-articular injection. In some aspects, the nucleic acid sequence encoding interleukin-10 has an amino acid substitution of the wild type phenylalanine at amino acid position 129, and in some aspects, the amino acid substitution is selected from the group of serine, alanine, threonine, or cysteine. F129S In yet another embodiment, the IL-10 expression construct comprises at least one nuclear targeting sequence 5' to the IL-10 coding sequence, and in some embodiments, the IL-10 expression construct comprises at least one nuclear targeting sequence 3' to the IL-10 coding sequence.

[0015] In some aspects of the invention, the microparticles comprise one or more of the following: poly(2-hydroxyethyl methacrylate), poly(N-vinylpyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid), polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), polyanhydrides, polycaprolactone, poly-3-hydroxybutyric acid, or polyorthoesters. In some aspects of the invention, the microparticles comprise PLGA. In other aspects of the invention, the microparticles comprise a mixture of biodegradable polymers that exhibit different complementary release characteristics.

[0016] In some embodiments of the method, the therapeutic anti-inflammatory IL-10 expression construct (and any adjuvant or "pretreatment" adjuvant) is delivered about every 40-120 days as needed for therapeutic effect, for example, for up to one year. In other embodiments, the therapeutic anti-inflammatory composition is delivered about every 40-120 days as needed for therapeutic effect for a period of more than one year. In yet other embodiments, the therapeutic anti-inflammatory composition is delivered about every 40-120 days as needed for therapeutic effect for the life of the subject.

[0017] The methods of the invention can also be used as a research tool to discover drugs, small molecules and / or biologics that can be used as "cocktails" together with therapeutic IL-10 expression construct compositions. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows results in bar graph form illustrating clinical assessment of functional improvement and pain relief achieved in treating osteoarthritis of the forelimb joints in dogs following administration of a therapeutic IL-10 expression construct of the invention. [Diagram 2] FIG. 1 shows results in bar graph form depicting owner assessment of improved function and pain relief achieved in treating osteoarthritis of the forelimb joints in dogs following administration of a therapeutic IL-10 expression construct of the invention. [Diagram 3] FIG. 1 shows results (pooled data) in bar graph form illustrating the clinical assessment of functional improvement and pain relief achieved in the treatment of osteoarthritis of the forelimb joints in dogs following administration of a therapeutic IL-10 expression construct of the invention. [Figure 4] FIG. 1 shows results (pooled data) in bar graph form illustrating owner assessment of improved function and pain relief achieved in treating osteoarthritis of the forelimb joints in dogs following administration of a therapeutic IL-10 expression construct of the invention. [Diagram 5] FIG. 1 shows results (pooled data) demonstrating the improvement in range of motion achieved in the treatment of osteoarthritis of the forelimb joints in dogs following administration of a therapeutic IL-10 expression construct of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, published patent applications, and patents mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing devices, animal models, formulations, and methods that can be used in connection with the invention described herein.

[0020] The term adjuvant as used herein refers to a pharmacological or immunological substance that modifies the effect of another drug. In the present case, adjuvants are used to enhance the efficacy of therapeutic IL-10 anti-inflammatory compounds. Adjuvants that are particularly useful in the present invention include those that increase the uptake or efficacy of IL-10 expression constructs by macrophages or other immune cells present in the synovial fluid of joints.

[0021] As used herein, the term "anti-inflammatory" refers to reducing the action or production of one or more pro-inflammatory cytokines or proteins produced by nerves, neurons, glial cells, endothelial cells, fibroblasts, muscle, immune cells, or other cell types.

[0022] As used herein, the term "anti-inflammatory cytokine" refers to a protein that reduces the action or production of one or more pro-inflammatory cytokines or proteins produced by nerves, neurons, glial cells, endothelial cells, fibroblasts, muscles, immune cells, or other cell types. Pro-inflammatory cytokines and proteins include, but are not limited to, interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and the like. Non-limiting examples of anti-inflammatory cytokines include interleukin-10 (IL-10), such as viral IL-10, interleukin-4 (IL-4), interleukin-13 (IL-13), α-MSH, transforming growth factor-β1 (TGFβ1), and the like. Thus, anti-inflammatory cytokines, as well as full-length molecules and fragments of anti-inflammatory cytokines having modifications such as deletions, additions and substitutions (either naturally conservative or non-conservative) to the native sequence are contemplated for use in the present invention, so long as the anti-inflammatory cytokine is therapeutically effective. Modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by host mutations generating the protein or errors resulting from PCR amplification. Thus, active proteins are typically substantially homologous to the parent sequence, e.g., proteins are typically about 70·80·85·90·95·98·99% homologous to the parent sequence.

[0023] An anti-inflammatory cytokine "coding sequence", i.e., a sequence that "encodes" an anti-inflammatory cytokine, is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by nucleotides corresponding to the start codon at the amino terminus and the translation stop codon at the carboxy terminus.

[0024] The term DNA "control sequences" collectively refers to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, enhancers, and the like, which collectively enable the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these types of control sequences need to be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.

[0025] The term "effective amount" or "therapeutically effective amount" of a therapeutic IL-10 expression construct used in the methods of the invention refers to a non-toxic but sufficient amount of the IL-10 expression construct to bring about the desired response, such as reducing joint inflammation, alleviating symptoms caused by inflammatory joint disease, and / or preventing the progression of joint damage caused by inflammatory joint disease. The exact amount required may vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the specific IL-10 expression construct to be delivered, whether the IL-10 expression construct is delivered in microparticles or as naked DNA, the method of administration (e.g., intra-articular injection), and the like. Dosage parameters for the methods are described herein; however, optimization of the appropriate "effective" amount in any individual case can be determined by one of skill in the art using the methods described herein and routine experimentation.

[0026] The term "excipient" or "diluent" refers to an inert substance added to a therapeutic composition of the invention to facilitate administration of a therapeutic IL-10 expression construct. Non-limiting examples of excipients include saline, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, hyaluronic acid (optionally formulated with a surfactant, Pluronic® F-68), vegetable oils and polyethylene glycols.

[0027] "Isolated," when referring to nucleotides, means that the expression construct is present in the substantial absence of other biological polymers of the same type. Thus, an "isolated nucleic acid molecule encoding a particular polypeptide" refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may contain some bases or moieties that do not adversely affect the basic characteristics of the composition.

[0028] The term "joint" refers to the anatomical structure where two bones meet, including the ligaments that connect the bones to one another, the tendons that attach muscles to the bones, the joint capsule, bursa, and synovial membrane. Joints that can be treated with the methods described herein include fixed, hinge, pivot, or ball and socket joints.

[0029] The term "arthritis" refers to all types of arthritis caused by inflammation (of which rheumatoid arthritis and osteoarthritis are the most common), as well as tendonitis, bursitis, inflammation of the ligaments, synovitis, gout, and systemic lupus erythematosus.

[0030] The term "nuclear targeting sequence" refers to a nucleic acid sequence that functions to improve the efficiency of expression of an anti-inflammatory cytokine in a cell. "Operably linked" refers to an arrangement of elements in which the elements so represented are configured to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting expression of the coding sequence. Control sequences need not be contiguous with the coding sequence, so long as they function to direct expression. Thus, for example, intervening sequences that are not translated, but are transcribed, can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0031] The term "promoter" is used herein in its ordinary sense to refer to a nucleotide region that contains DNA regulatory sequences, where the regulatory sequences are derived from a gene capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. Transcriptional promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter can be induced by a test substance, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter can be induced by a test substance, cofactor, regulatory protein, etc.), and "constitutive promoters."

[0032] Throughout this application, for example, when a particular nucleotide sequence is described as being located "upstream," "downstream," "three dashes (3')," or "five dashes (5')" relative to another sequence, for purposes of describing the relative position of a nucleic acid sequence in a particular nucleic acid molecule, this should be understood to be the location of the sequence on the "sense" or "coding" strand of the DNA molecule, as commonly referred to in the art.

[0033] As used herein, the term "research tool" refers to any method of the invention that employs a therapeutic IL-10 expression construct for scientific investigations of either an academic or commercial nature, including the development of other pharmaceutical and / or biological therapeutics. The research tools of the invention are not intended to be therapeutic or to be approved by regulatory agencies; rather, the research tools of the invention are intended to facilitate research and support in development activities, such as any activities conducted for the purpose of generating information in support of a submission.

[0034] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal. As used herein, the term "therapeutic composition" or "therapeutic anti-inflammatory composition" refers to a composition having the ability to reduce joint inflammation, provide relief from symptoms caused by inflammatory joint diseases, and / or inhibit the progression of joint damage resulting from inflammatory joint diseases, as measured in any of the known animal models or by assessments performed in humans.

[0035] "Treatment" or "treating" arthritis includes: (1) reducing joint inflammation or causing inflammation to occur at a lower intensity in a subject believed to be predisposed to arthritis but not yet experiencing or exhibiting symptoms, or (2) inhibiting arthritis, i.e., halting or reversing the development of symptoms or physiological damage caused by inflammation.

[0036] As used herein, a "viral vector" is a recombinant virus or viral particle that contains an IL-10 expression construct to be delivered to a host cell, either in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, alphaviral vectors, and the like.

[0037] The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry, and sequencing techniques that are within the skill of those of ordinary skill in the art. Specific illustrations of suitable techniques may be had by reference to the examples described herein. However, it will be appreciated that other equivalent general procedures may also be used. Such prior techniques and descriptions can be found, for example, in standard laboratory manuals such as: LeDoux (ed.) (2005), Animal Models of Movement Disorders (Academic Press); Chow et al. (2008), Using Animal Models in Biomedical Research (World Scientific Publishing Co.); Weir and Blackwell (eds.), Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications; Creighton (1993), Proteins: Structures and Molecular Properties (WH Freeman & Co., 2004).Freeman and Company); Sambrook and Russell (2006), Condensed Protocols from Molecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002), Molecular Cloning: A Laboratory Manual (both from Cold Spring Harbor Laboratory Press); Stryer, L. (1995), Biochemistry, 4th ed. (WH Freeman); Gait (1984), Oligonucleotide Synthesis: A Practical Approach: (IRL Press) Press); Nelson and Cox (2000), Lehninger, Principles of Biochemistry, 3rd Edition (WH Freeman); and Berg et al. (2002), Biochemistry, 5th Edition (WH Freeman); all of which are incorporated by reference in their entirety for all purposes.

[0038] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0039] When a range of values ​​is stated, it is understood that each intervening value between the upper and lower limits of that range, as well as any other stated or intervening value within the stated range, is included in the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, but are also included in the invention, subject to any limit specifically excluded in the stated range. When a stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0040] In the following description, numerous details are set forth to facilitate a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without one or more of these details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the present invention.

[0041] Methods of the Invention The present invention provides a method for treating inflammatory joint diseases, symptoms associated with inflammatory joint diseases, and slowing disease progression by administering to a subject, typically by injection, a vector expressing a therapeutic interleukin-10 (IL-10) expression construct. In some embodiments, the IL-10 expression construct is encapsulated in biodegradable microparticles, and in most aspects of this embodiment, the microparticles are suspended in a diluent to form a therapeutic composition. Alternatively, in a preferred embodiment, the therapeutic IL-10 expression vector is delivered as a "naked" vector, where delivery of the naked IL-10 expression vector is performed together with or after administration of a nucleic acid uptake adjuvant. The nucleic acid uptake adjuvant may be administered either simultaneously with administration of the naked IL-10 expression vector, or up to 10 days or more prior to its administration. The method of the present invention may be used to treat arthritis in the knee, elbow, wrist, ankle, hip, shoulder, or spine. Conditions treatable by the methods of the invention include rheumatoid arthritis and osteoarthritis, as well as tendonitis, bursitis, ligament inflammation, synovitis, gout, and systemic lupus erythematosus.

[0042] Thus, the present invention generally provides methods for treating inflammatory joint diseases, as well as symptoms or physiological damage associated with inflammatory joint diseases. The present invention also provides the use of the methods of the present invention to study inflammatory joint diseases, such as to find drugs, small molecules and / or biologics that can be used together with a therapeutic IL-10 expression construct (as a "cocktail"). The methods of the present invention include administering to a subject (preferably by intra-articular injection) an IL-10 expression construct comprising a bacterial or viral scaffold and at least one IL-10 coding sequence. In some embodiments, the IL-10 expression construct is optionally encapsulated in biodegradable microparticles. The anti-inflammatory composition is generally suspended in a diluent for delivery to the joint. The IL-10 expression construct may include at least one nuclear targeting sequence, where the at least one nuclear targeting sequence is 5', 3' or both 5', 3' or both sides of the IL-10 coding sequence.

[0043] The anti-inflammatory compositions of the invention may be comprised of a "naked" bacterial or viral vector capable of expressing at least one IL-10 coding sequence, or alternatively, the anti-inflammatory compositions may be comprised of a bacterial or viral vector encased in a microparticle or other delivery device. Administration of the therapeutic IL-10 expression construct may occur together with or after administration of an adjuvant, and in the case of delivery of a naked IL-10 expression construct, the nucleic acid incorporating adjuvant may be administered simultaneously with or up to 10 days or more prior to administration of the naked IL-10 expression construct.

[0044] The IL-10 expression construct used in some embodiments of the method of the present invention comprises a bacterial backbone (plasmid DNA or pDNA) or a viral backbone, at least one IL-10 coding sequence, at least one nuclear targeting sequence 5' (upstream), 3' (downstream) or both of the at least one IL-10 coding sequence, and one or more DNA regulatory sequences. Optionally, the pDNA may also comprise one or more additional anti-inflammatory cytokine coding sequences, and / or a marker sequence that allows for the selection of transformed cells during amplification of the pDNA. The bacterial backbone may be any bacterial backbone known to those skilled in the art. Typically, the backbone selected will be, for example, one that contains or lacks suitable restriction sites to facilitate cloning, one that can be easily produced and isolated, one that is not immunogenic, etc. For example, bacterial backbones derived from E. coli are useful in the present invention.

[0045] The plasmid DNA comprises at least one IL-10 coding sequence. The IL-10 coding sequence may encode wild-type IL-10 or the IL-10 may be a mutant IL-10. One mutant IL-10 of interest comprises one or more mutations resulting in amino acid substitutions, additions or deletions in the "hinge" region of the IL-10 protein compared to wild-type IL-10. The human IL-10 protein is a homodimer, with each monomer comprising six α-helices A→F, the lengths of which are 21, 8, 19, 20, 12 and 23 amino acids, respectively. Helix A→F of one monomer non-covalently interacts with helices E and F of the second monomer to form a non-covalent V-type homodimer. In accordance with the present invention, the "hinge" region targeted for mutation comprises amino acids between the D and E alpha helices on one or both monomers, from about amino acid position X to position Y of wild-type IL-10. For example, mutant rat and human IL-10 proteins have been described in which the phenylalanine at position 129 of the wild-type sequence is replaced with a serine residue. (See, e.g., Sommer et al., WO 2006 / 130580; and Milligan et al., Pain 126:294-308 (2006)). The resulting mutant IL-10 has the same IL-10 activity as the wild-type IL-10. F129S Other substitutions for the wild-type phenylalanine at amino acid position 129 may be, for example, threonine, alanine, or cysteine. Thus, the invention in further embodiments includes one or more substitutions at amino acid position 129 or other amino acids within the hinge region of the IL-10 protein, or functional equivalents thereof.

[0046] Additional anti-inflammatory cytokines useful in the present invention include, but are not limited to, interleukin-4 (IL-4), interleukin-13 (IL-13), α-MSH, transforming growth factor-β1 (TGFβ1), and the like.

[0047] The nuclear targeting sequence of the present invention is a sequence that promotes the expression of the protein encoded by at least one IL-10 coding sequence and optional another anti-inflammatory cytokine coding sequence.For example, in one aspect, the nuclear targeting sequence binds to nuclear transport chaperone proteins to promote the uptake of plasmid DNA by the cell nucleus.Such sequences include, but are not limited to, interspersed (or dispersed) DNA repeats or repeat sequences such as transposable elements, flanking or terminal repeats such as long terminal repeats (LTRs) on retroviral genomes such as SV40, tandem repeats, and inverted terminal repeats (ITRs) of viral genomes such as Adeno-Associated Virus and Adenovirus.In another aspect, the nuclear targeting sequence is a sequence that acts to bind transcription factors for import into the nucleus, such as an enhancer sequence.

[0048] In addition to the bacterial backbone, at least one IL-10 coding sequence, and optionally one or more additional anti-inflammatory cytokine coding sequences, and optionally one or more nuclear targeting sequences, the plasmid DNA of the present invention includes one or more DNA control sequences, such as a promoter sequence, a polyadenylation signal, a transcription termination sequence, an upstream regulatory domain, an origin of replication, an internal ribosome entry site, and the like, which collectively allow for the replication, transcription, and translation of the anti-inflammatory cytokine coding sequence in the recipient cell. It is not necessary that all of these sequences are always present, so long as the anti-inflammatory cytokine coding sequence can be replicated, transcribed, and translated in an appropriate host cell. Promoter sequences useful in the present invention include, but are not limited to, chicken or human β-actin promoter, cytomegalovirus immediate early promoter, glyceraldehyde 3-phosphate dehydrogenase (GADPH) promoter, elongation factor 1 alpha (eF1α) promoter, GFAP promoter, murine leukemia virus (MLV) promoter, herpes simplex virus thymidine kinase (TK) promoter, and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promoter; upstream regulatory domains useful in the present invention include, but are not limited to, cytomegalovirus immediate early promoter enhancer, mouse mammary tumor virus (MMTV) enhancer, and simian virus 40 (SV40) enhancer; polyadenylation signals of interest in the present invention include, but are not limited to, SV40 polyadenylation signal, bovine growth hormone polyadenylation signal, and synthetic polyadenylation signals. Optionally, the plasmid DNA of the present invention also contains a selectable marker gene, such as one encoding antibiotic resistance. Marker genes useful in the present invention include, but are not limited to, neomycin, hygromycin-B, ampicillin, kanamycin, or puromycin.

[0049] For example, a plasmid containing the following: a rat IL-10 sequence flanked by two AAV ITRs, a cytomegalovirus immediate early promoter enhancer, a chicken β-actin promoter, a polyadenylation signal, and a herpes simplex thymidine kinase promoter driving a neomycin resistance marker was used in some experiments to demonstrate the utility of the present invention. In other experiments, a plasmid containing the following: a human IL-10 sequence flanked by two AAV ITRs, a cytomegalovirus immediate early promoter enhancer, a cytomegalovirus immediate early promoter, a polyadenylation signal, and an ampicillin resistance marker was used. Details of these plasmids are disclosed in Milligan et al., Pain, vol. 126, pp. 294-308 (2006).

[0050] Alternatively, the vector may be a viral vector. In general, the five most commonly used classes of viral systems used in gene therapy can be divided into two groups, depending on whether their genomes are integrated into host cell chromatin (oncoretroviruses and lentiviruses) or persist in the cell nucleus as mostly extrachromosomal episomes (adeno-associated viruses, adenoviruses, herpesviruses, and integration-defective lentiviruses). For example, in one embodiment of the present invention, a virus from the Parvoviridae family is used. The Parvoviridae family is a family of small, single-stranded, non-enveloped DNA viruses with genomes of about 5000 nucleotides in length. Members of this family include adeno-associated viruses (AAV), i.e., dependent parvoviruses that, by definition, require co-infection with another virus (typically adenovirus or herpesvirus) to initiate and maintain a productive infection cycle. Even in the absence of such helper viruses, AAV remains capable of infecting or transducing target cells through receptor-mediated binding and internalization, and of inserting into the nuclei of both non-dividing and dividing cells.

[0051] Another viral delivery system useful with the IL-10 expression constructs of the present invention is a system based on viruses from the Retroviridae family. Retroviruses include single-stranded RNA animal viruses that are characterized by two unique features. First, the genome of retroviruses is diploid, consisting of two copies of RNA. Second, this RNA is transcribed into double-stranded DNA by the virion-associated enzyme reverse transcriptase. This double-stranded DNA, or provirus, can then be integrated into the host genome and passed from parental cells to progeny cells as a stably integrated component of the host genome.

[0052] In some embodiments, lentiviruses are preferred members of the retrovirus family for use in the present invention. Lentivirus vectors are often pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) and are derived from human immunodeficiency virus (HIV), the etiological agent of human acquired immune deficiency syndrome (AIDS); visan-maedi, which causes encephalitis (visna) or pneumonia in sheep; equine infectious anemia virus (EIAV), which causes autoimmune hemolytic anemia and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immunodeficiency in cats; bovine immunodeficiency virus (BIV), which causes lymphadenopathy and lymphocytosis in cattle; and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalopathy in non-human primates. HIV-based vectors generally retain <5% of the parental genome and incorporate <25% of the genome into the packaging construct, minimizing the possibility of generating reverted replication-competent HIV. Biosafety has been further enhanced by the development of self-inactivating vectors that contain deletions of regulatory elements in the downstream long terminal repeats, eliminating transcription of the packaging signal required for vector mobilization. The main advantage of using lentiviral vectors is that gene transfer is persistent in most tissues or cell types due to integration of the viral vector. However, integrase-deficient lentiviruses can also be used.

[0053] Adenoviruses (Ad) are a relatively well-characterized homogeneous group of viruses, including more than 50 serotypes. See, for example, International PCT Application WO 95 / 27071. Adenoviruses are medium-sized (90-100 nm), non-enveloped (without an outer lipid bilayer) icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. In humans, 57 serotypes have been described, which are responsible for 50-10% of upper respiratory tract infections in children and many infections in adults. They are classified as group I according to the Baltimore classification scheme, which means that their genome consists of double-stranded DNA and are the largest non-enveloped viruses. Due to their large size, they can be transported through endosomes (i.e., no envelope fusion is required). The virion also has unique "spikes" or fibers associated with each penton base of the capsid, which aid in binding to host cells via the Coxsackie-Adenovirus receptor on the surface of the host cell.

[0054] The adenovirus genome is a linear, non-segmented, double-stranded (ds) DNA, 26-45 kb, allowing the virus to theoretically carry 22-40 genes. This is significantly larger than other viruses in its Baltimore group, but it is still a very simple virus and is highly dependent on the host cell for survival and replication. Once the virus successfully enters the host cell, the endosomes acidify, which modifies the viral topology by dissociating the capsid components. With the help of cellular microtubules, the virus is transported to the nuclear pore complex, where the adenovirus particle disassembles. Subsequently, the viral DNA is released, which can enter the nucleus through the nuclear pore. The DNA then binds to histone molecules. Thus, viral gene expression can occur and new viral particles can be generated.

[0055] Unlike most lentiviral vectors (i.e., capable of integration, e.g., not integrase-deficient), adenoviral DNA is not integrated into the genome and is not replicated during cell division. The main applications of adenoviruses are in gene therapy and vaccination. Recombinant adenovirus-derived vectors have also been constructed, particularly those that reduce the capacity for recombination and production of wild-type virus. See International PCT Publication Nos. WO 95 / 00655 and WO 95 / 11984.

[0056] Other viral or non-viral systems known to those skilled in the art can be used to deliver the IL-10 expression constructs of the invention to the joint, including, but not limited to, gene deleted adenovirus-transposon vectors that stably maintain the virus encoded transgene in vivo upon integration into the host cell (see Yant et al., Nature Biotech. 20:999-1004 (2002)); systems derived from Sindbis virus or Semliki forest virus (see Perri et al., J. Virol. 74:20:9802-07 (2002)); systems derived from Newcastle disease virus or Sendai virus; or minicircle DNA vectors lacking bacterial DNA sequences (Chen et al., Molecular Therapeutics 20:131-135 (2002)). Therapy, Vol. 8, No. 3, pp. 495-500 (2003). The minicircle DNA described in U.S. Patent Application Publication No. 2004 / 0214329 discloses a vector that provides sustained high-level nucleic acid transcription.

[0057] Once the IL-10 expression construct has been constructed, amplified and isolated by techniques known in the art, in some embodiments, the IL-10 expression construct is optionally encapsulated in a microparticle. The technique for encapsulating the IL-10 expression construct varies depending on the type of microparticle used, and such techniques are described in detail below. The microparticles of the present invention may be composed of any biodegradable polymer. To be successfully used as a biodegradable polymer in the controlled drug delivery formulations of the present invention, the material must be chemically inert and free of leachable impurities. Ideally, the polymer also has the appropriate physical structure, exhibits minimal undesirable aging, and is easily processable. Some of the materials include poly(2-hydroxyethyl methacrylate), poly(N-vinylpyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), and poly(methacrylic acid). Particularly useful biodegradable polymers in the present invention include polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), polyanhydrides, polycaprolactone, poly-3-hydroxybutyric acid, and polyorthoesters. Such biodegradable polymers have been extensively characterized and can be formulated to exhibit the desired degradation characteristics, as is known in the art (see, e.g., Edlund and Albertsson, Degradable Aliphatic polyesters, 67-112 (2002); Barman et al., J. of Controlled Release, 69:337-344 (2000); Cohen et al., Pharmaceutical Res., 8:713-720 (1991)).

[0058] In one specific embodiment of the present invention, the polymer comprises poly(lactide-co-glycolide) (PLGA). PLGA is a copolymer that has been used in matrices of FDA approved therapeutic devices due to its biodegradability and biocompatibility. PLGA is synthesized using a cyclic dimer of glycolic acid and lactic acid (1,4-dioxane-2,5-dione), which is a random ring-opening copolymerization of two different monomers. Common catalysts used in the preparation of this polymer include tin(II) 2-ethylhexanoate, tin(II) alkoxides, or aluminum isopropoxide. During polymerization, consecutive monomer units of glycolic acid or lactic acid are linked together in PLGA by ester bonds, thus resulting in a linear aliphatic polymer as the product.

[0059] Depending on the ratio of lactide and glycolide used in the polymerization, different forms of PLGA can be obtained; these are usually distinguished with respect to the ratio of monomers used (e.g., PLGA 75:25 indicates a copolymer whose composition is 75% (mol percent) lactic acid and 25% (mol percent) glycolic acid). PLGA degrades by hydrolysis of its ester bonds in the presence of water. The time required for degradation of PLGA is related to the ratio of monomers used in its production: the higher the content of glycolide units, the shorter the time required for degradation is shown. An exception to this rule is a copolymer with a 50:50 monomer ratio, which exhibits a faster degradation (about 2 months). Furthermore, polymers end-capped with esters (as opposed to free carboxylic acids) show a longer degradation half-life. Particularly useful in the present invention are PLGAs with a composition of 20%-80% lactic acid and 80%-20% glycolic acid. More useful in the present invention is PLGA having a composition of 65% to 35% lactic acid and 35% to 65% glycolic acid. In one embodiment of the present invention, PLGA having a composition of 50% lactic acid and 50% glycolic acid is used.

[0060] Additionally, the IL-10 expression construct (pDNA) may be encapsulated into a batch of microparticles with different release profiles; for example, 10% of the pDNA to be delivered may be encapsulated in microparticles with a release profile of, for example, 1 day to 4 weeks; 30% of the pDNA to be delivered may be encapsulated in microparticles with a release profile of, for example, 3 weeks to 6 weeks; 30% of the pDNA to be delivered may be encapsulated in microparticles with a release profile of, for example, 6 weeks to 10 weeks; 30% of the pDNA to be delivered may be encapsulated in microparticles with a release profile of, for example, 8 weeks to 12 weeks. In such an embodiment, a single type of biodegradable polymer may be used, but this may be used in formulations with different release profiles; alternatively, different biodegradable polymers with different release characteristics may be used. In yet another embodiment, the formulation of the microparticles may be modified to change the surface of the microparticles, for example, to enhance or retard the movement of the therapeutic composition from the synovial fluid of the joint, as required.

[0061] Once the microparticles are obtained, they are suspended in an acceptable diluent to form a therapeutic composition for administration to a subject. Similarly, when the IL-10 expression construct of the present invention is delivered as naked DNA as opposed to encapsulation, a diluent is also used for administration to a subject. Such diluents (or excipients) include any agent that does not itself induce the production of antibodies harmful to the individual receiving the composition and that can be administered without undue toxicity. Pharmaceutically acceptable diluents may include sorbitol, alum, dextran, sulfates, high molecular weight polymeric anions, any of the various TWEEN® compounds, liquids such as water, saline, glycerol or ethanol, oils and aqueous emulsions, or adjuvants such as Freund's adjuvant. Pharmaceutically acceptable salts may also be included, such as, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids such as acetate, propionate, malonate, benzoate, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, etc., may be present in such vehicles. In one aspect of the invention, various diluents are used depending on their ability to migrate from the target site in the joint. For example, for delivery to adult humans, a diluent that promotes more rapid spreading of the therapeutic composition from the joint may be preferred; conversely, for children or small animals where the size of the joint is less critical, a diluent that does not disperse the therapeutic composition as rapidly may be used. A comprehensive description of pharma- ceutically acceptable diluents / excipients is available in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams and Wilkins (2005). Preferred diluents include, but are not limited to, Physiosol®, artificial synovial fluid, preservative-free 0.9% NaCl, Lactated Ringer's Injection, and Elliotts B® Solution.

[0062] The synthesis and administration of the compositions used in the methods of the invention involves a series of steps. First, a plasmid is constructed containing the various components described above. Then, the plasmid DNA (pDNA) is amplified and isolated by techniques known in the art. Once the pDNA is isolated, it can be combined with a polymer to form pDNA-containing microparticles. The method of microparticle formation varies depending on the polymer used, but typically uses a two-phase emulsion method. First, the polymer is dissolved in an organic solvent. Next, the pDNA is suspended in an aqueous solution and then added to the polymer solution. The two solutions are then mixed to form a first emulsion. The solutions can be mixed by vortexing or shaking, or passing through a granular medium that creates turbulence, or the mixture can be sonicated. Most preferred is any method that allows for the formation of a suitable emulsion while at the same time incurring the least amount of damage to the nucleic acid in the form of nicking, shearing, or degradation. At this step, the polymer is formed into microparticles, many of which contain pDNA. If desired, a small amount of the nucleic acid can be isolated at this point to assess its integrity, for example, by gel electrophoresis.

[0063] The first emulsion is then added to an organic solution. The solution can be composed of, for example, methylene chloride, ethyl acetate, or acetone, and typically contains polyvinyl alcohol (PVA), often with a ratio of about 1:100 weight of PVA to volume of solution. The first emulsion is generally added to the organic solution while stirring in a homogenizer or ultrasonicator. In this step, a second emulsion is formed, which is then added to another organic solution while stirring (e.g., in a homogenizer). In one embodiment of the method, the latter solution is 0.05% w / v PVA. The resulting microparticles are washed several times with water to remove organic compounds. In some embodiments of the invention, more than about 40% of the resulting microparticles contain pDNA. In other embodiments, more than about 50% of the resulting microparticles, and in yet other embodiments of the invention, more than about 55% of the resulting microparticles contain pDNA.

[0064] Different cell types have different abilities to internalize microparticles of different sizes. In some embodiments of the present invention, macrophages and antigen presenting cells were targeted. Such cells internalize microparticles less than about 5μ more efficiently (see Shakweh et al., Eur J of Pharmaceutics and Biopharmaceutics, vol. 61, no. 1-2, pp. 1-13 (2005)). Therefore, if necessary, the particles can be sieved to selectively remove particles larger than the desired size. In one specific embodiment of the present invention, microparticles less than 5μ are used in the therapeutic composition, and in another specific embodiment of the present invention, microparticles less than 3μ are used in the therapeutic composition. After washing, the particles can be used immediately or lyophilized for storage. The size distribution of the microparticles prepared by the methods described herein can be determined, for example, by a Coulter™ counter or laser diffraction. Alternatively, the average particle size of the particles can be determined by visualization under a microscope equipped with a sizing slide or eyepiece. Alternatively, scanning electron microscopy can be used to assess both particle size and microparticle morphology.

[0065] Once the IL-10 expression construct-containing microparticles are obtained, they can be immediately suspended in a diluent or lyophilized for storage. The combination of the microparticles and the diluent forms a therapeutic microparticle composition that can be administered to a subject animal by injection into a joint. The recombinant vector can be introduced either in vivo or in vitro (also called ex vivo) to treat arthritis. When transducing in vitro, the desired recipient cells or synovial fluid are taken from the subject, treated with the pDNA-containing microparticles, and reintroduced into the subject. Alternatively, syngeneic or xenogeneic cells can be transformed for delivery, if they generally do not generate an inappropriate immune response in the subject. When administered in vivo, the recombinant vector, or cells transformed with the vector in vitro, are delivered directly by injection into the joint.

[0066] In another embodiment, the IL-10 expression construct of the present invention is administered as naked DNA. In such an embodiment, for example, the IL-10 expression construct is amplified using good manufacturing practices. GMP is enforced in the United States by the Food and Drug Administration (FDA) under Section 501(B) of the Food, Drug, and Cosmetic Act of 1938 (21 USCS §351).

[0067] Adjuvants suitable for the present invention include adjuvants that enhance uptake of the IL-10 expression constructs of the present invention; i.e., adjuvants suitable for the present invention include any biocompatible agent that overcomes or avoids the problem of introducing negatively charged DNA into cells with negatively charged membranes. Such adjuvants include sugars such as mannose, glucose and sucrose; calcium phosphate; dendrimers (repeatedly branched molecules); liposomes (spherical vesicles containing lipid bilayers), such as cationic liposomes; DEAE-dextran, such as DEAE-dextran polyethyleneimine; oligodeoxynucleotides; and high molecular weight hyaluronic acid (>1 MDa), an anionic non-sulfated glycosaminoglycan.

[0068] One particularly useful adjuvant is D-mannose, a simple hexose sugar with a molecular weight of 180.2 that reduces inflammatory processes during wound healing (Kossi J et al., Eur Surg Res 31(1):74-82 (1999)), reduces the oxidative burst required during inflammation (Rest RF et al., J Leukoc Biol 43(2):158-164 (1988)), and inhibits adjuvant-induced arthritis in a rat model (Willenborg DO et al., Immunol Cell Biology 1999:111-112 (1999)). Biol, vol. 70, no. 6, pp. 369-377 (1996)), inhibit LPS-induced IL-1β, TNF-α, reduce NF-kB / p65, which is crucial for proinflammatory cytokine expression, and reduce leukocyte influx after intratracheal instillation of LPS, a model of sepsis-associated acute lung injury and respiratory distress syndrome (Xu XL et al., Inflamm Res, vol. 57, no. 3, pp. 104-110 (2008); Xu X et al., Eur J Pharmacol, vol. 641, no. 2-3, pp. 229-237 (2010)). MR is a transmembrane glycoprotein pattern recognition that participates in innate immune host defense by recognizing mannosylated ligands (e.g., lysosomal hydrolases), which may include a variety of bacteria, yeast, and parasites that express mannosylated molecules (see, e.g., Engering AJ et al., Adv Exp Med Biol 417:183-187 (1997); Linehan SA et al., Adv Exp Med Biol 417:183-187 (1997)). Med Biol, 479, 1-14 (2000); Stahl PD et al., Curr Opin Immunol, 10, 1, 50-55 (1998).

[0069] The therapeutic compositions of the present invention do not significantly induce immune responses or dose tolerance in the subject and can therefore be administered as needed for therapeutic efficacy. That is, the therapeutic anti-inflammatory composition can be delivered about every 40-120 days (or as needed) for short-term therapy as needed for therapeutic efficacy. However, if longer-term therapy is desired, the therapeutic composition can be delivered about every 40-120 days (or thereabouts) as needed for therapeutic efficacy for periods greater than one year; for the life of the subject, if necessary. The frequency of administration will vary depending on the dosage, the adjuvant used, and the health of the subject.

[0070] Dosage ranges for the therapeutic compositions used in the methods of the invention will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the site of the joint, and the specific IL-10 expression construct to be delivered, whether the IL-10 expression construct is encapsulated, the method of administration, etc. Dosage ranges include a therapeutically effective amount of about 1-1000 μg vector DNA, about 5-750 μg vector DNA, about 10-600 μg vector DNA, 20-500 μg vector DNA, 25-250 μg vector DNA, or 50-100 μg vector DNA per joint.

[0071] The IL-10 expression constructs, or microparticles containing an IL-10 expression construct, used in the methods of the invention may be co-administered as a "cocktail" with other therapeutic agents useful in the treatment of arthritis, including glucocorticoids: methotrexate; hydroxychlorquine; sulfasalazine; lefunomide; anti-TNF agents such as etanercept, infliximab and adalimumab; abatacept; hyaluronic acid, particularly high molecular weight hyaluronic acid (>1 MDa), such as Hyalgan, Orthovisc, or Synvisc, at a dose of 1-5 mL per joint, thus 5 mg to 125 mg, for example, 0.5 to 2.5% (5 to 25 mg / mL); and non-steroidal anti-inflammatory drugs (NSAIDs). Additionally, the IL-10 expression construct or microparticles containing an IL-10 expression construct used in the methods of the invention may be co-administered with cells, such as mesenchymal stem cells or other stem cells, including stem cells genetically modified to express the IL-10 expression construct. Generally, any method known in the art can be used to monitor the success of treatment in humans, including both clinical and phenotypic indicators.

[0072] Additional objects, advantages, and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples, which are not intended to be limiting, in which procedures that have been reduced to practice are described in the present tense and procedures that were performed in the laboratory are described in the past tense.

[0073] (Example) The following examples are provided to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent or imply that the experiments presented below are all or the only experiments performed. Those skilled in the art will appreciate that various changes and / or modifications may be made to the present invention without departing from the spirit or scope of the invention as broadly described, as shown in the specific embodiments. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0074] Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0075] Amplification and purification of pDNA The plasmid construct encoding rat interleukin-10 (pDNA-IL-10F129S) has been previously described in detail by Milligan et al., Pain, vol. 126, no. 1-3, pp. 294-308 (2006). Briefly, the plasmid consists of a 5.9 kilobase (Kb) circular plasmid DNA containing a transcription cassette consisting of the cytomegalovirus enhancer / chicken β-actin promoter (CMV enh / CB pro) driving expression of the rat IL-10 gene containing a point mutation (F129S) and a viral SV40 polyadenylation signal. The transcription cassette is flanked by 149 bp inverted terminal repeats. An identical plasmid lacking IL-10 was used as a pDNA control. Both plasmids were expressed in the SURE The plasmid was amplified in 2 supercompetent E. coli cells (Agilent Technologies, USA) and isolated using the Endotoxin-Free Giga Plasmid Purification Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The purified endotoxin-free plasmid was resuspended in sterile Dulbecco's PBS (DBPS, 1, 0.1 micron pore filtered, pH 7.2, Cat# 14190-144, Gibco, Invitrogen Corp, Grand Island, NY, USA) containing 3% sucrose (DPBS-3%). The DPBS-3% vehicle was prepared using molecular biology grade D(+)-sucrose (bD-fructofuranosyl-aD-glucopyranoside; Sigma-Aldrich, St. Louis, MO, USA) in DPBS and stored sterile in 15 ml conical tubes at 4°C until use after 0.2 um sterile filtration (pyrogen-free syringe filter unit, catalog #25AS020AS, Life Science Products, Inc., CO, USA).

[0076] Microparticle preparation and characterization Microparticles were prepared using a modified two-phase emulsion / solvent evaporation protocol (AM Tinsley-Brown et al., J. of Controlled Release, vol. 66, no. 2-3, pp. 229-41 (2006)). Briefly, 50:50 PLGA copolymer (MW 75,000, Lactel Absorbable Polymers) was dissolved in ethyl acetate (Sigma). Vehicle alone (phosphate buffered saline (PBS) + 3% (w / v) sucrose (Sigma)) or pDNA in vehicle was emulsified in PLGA solution and then re-emulsified in 5% (w / v) polyvinyl alcohol, 28% calcium chloride, 3% sucrose (Sigma) and 7% (v / v) ethyl acetate solution. After 4 hours of hardening in the washing solution, the resulting microparticles were collected, lyophilized, and stored at 4°C. Scanning electron microscopy (SEM) was used to examine the morphology of the microparticles. Microparticles with a particle size >1000 present in 10 different images were measured by NIH ImageJ software, and normalized frequency distributions were generated using binned particle sizes. The endotoxin levels of the resulting microparticles were tested by LAL assay using serial dilutions as inhibition controls after measuring the zeta potential of the microparticles by Nicomp 380 ZLS Zeta Potential Analyzer. The microparticles used showed a spherical smooth morphology under SEM, with a zeta potential of -28.04 ± 2.12 mV. The microparticles showed a heterogeneous particle size distribution with an overall median particle size of 4.67 ± 0.26 μm, which is consistent with the similar microparticle manufacturing method, and the pDNA encapsulation efficiency of the particles was 55.1%.

[0077] Total pDNA encapsulation was assessed by extracting pDNA from the microparticles by sodium hydroxide dissolution, measuring absorbance at 260 nm, and comparing the obtained values ​​to DNA standards of known concentration. Final pDNA loading was 8.78±0.65 μg pDNA / mg PLGA for PLGA-pDNA-IL-10 microparticles. Aqueous extraction of pDNA was performed by dissolving the microparticles in chloroform and running the pDNA in aqueous buffer. The extracted pDNA was then concentrated by precipitation with ethanol before being resuspended in PBS+3% sucrose vehicle. The structural integrity of the aqueous extracted pDNA was compared to non-encapsulated pDNA (which was also subjected to the aqueous extraction step) by loading 2 μg of total pDNA into the wells of a 1.0% agarose gel containing ethidium bromide, running the gel at 75 V for 2 hours, and imaging the gel by UV transillumination at 305 nm. The biological activity of aqueous extracted pDNA was assessed by lipofectamine-mediated transfection into human embryonic kidney 293 cells according to the manufacturer's protocol (Invitrogen), after which IL-10 protein concentrations in cell culture supernatants harvested 24 hours after aqueous extraction and transfection with non-encapsulated pDNA were assessed by ELISA (R&D Systems). In vitro release profiling was performed by incubating the microparticles in PBS over time in a water bath at 37°C, after which pDNA content in the supernatants was quantified by PicoGreen assay (Milligan et al., Neuron Glia Biology 2:4:293-308 (2006)).

[0078] Agarose gel electrophoresis of aqueous extracted pDNA from microparticles compared to non-encapsulated pDNA showed that although little detection of linearized pDNA and some changes in migration of multimeric pDNA species were observed after encapsulation, a significant amount of relaxation and integrity of supercoiled pDNA structure was preserved after encapsulation. Comparing IL-10 protein expression levels obtained in the supernatant of human embryonic kidney 293 cells with dose-matched microparticle-extracted or non-encapsulated pDNA 24 hours after lipofectamine-mediated transfection, it was found that microparticle-extracted pDNA-IL-10 showed 96.8% bioactivity retention for the resulting production of IL-10 (data not shown). In vitro pDNA release analysis demonstrated that 30% of the pDNA was released after 3 days, with a stable release achieved over a period of more than 75 days. This two-phase release profile is a common feature of macromolecule release from emulsion-based PLGA microparticles, with an initial enhanced pDNA release phase due to increased pDNA content on or near the surface of the microparticles, followed by sustained release and diffusion of pDNA from the interior of the microparticles (Yeo, Archive of Endotoxin Research, 2011). Res., Vol. 27, No. 1, pp. 1-12 (2004). Endotoxin levels from microparticles with or without encapsulated pDNA were below the detection limit for the LAL assay at or below a microparticle concentration of 10 mg / ml (1 mg of microparticles / well).

[0079] D-Mannose In embodiments using an adjuvant such as D-mannose, D-mannose (catalog #M6020) can be purchased carrier free from Sigma-Aldrich (St. Louis, MO). D-mannose is combined with 0.1% BSA in sterile saline and administered by intra-articular injection either simultaneously with the IL-10 expression construct or 1-10 days prior to administration of the IL-10 expression construct. D-mannose is typically delivered at a dose of 2.5 μg to 500 μg per joint in dogs.

[0080] Administration for the treatment of arthritis in dogs IL-10 expression construct as "naked" pDNA (pDNA-IL-10 F129S ) was administered by acute intra-articular injection into the affected joint of a group of dogs. Under intravenous sedation and anesthesia monitoring, the affected joint of the canine patient was surgically shaved to remove all hair over the affected joint and sterilized using a surgical scrub such as Chlorahexidine 2%. The patient's heart rate, blood pressure, oxygen saturation, ventilation, and cardiac rhythm were continuously monitored. A 22 or 20 gauge hypodermic needle was inserted into the synovial cavity. To ensure proper placement of the needle, synovial fluid was aspirated prior to administration of the pDNA. The syringe containing the synovial fluid aspirate was replaced while maintaining needle placement within the joint. Once the synovial fluid was aspirated, the therapeutic anti-inflammatory composition was administered into the joint using the same intra-articular needle. A maximum of 1 mg of the equivalent of plasmid DNA was injected, although as little as 700 μg was found to be effective. During administration, joint synovial fluid was aspirated to balance if the amount per joint injection exceeded 1 ml. After successful introduction of the therapeutic anti-inflammatory composition into the joint cavity, the patient was reversed from sedation and clinically monitored. Any changes to blood pressure, heart rate, oxygen administration, ventilation, or cardiac rhythm during the procedure were corrected with appropriate medical measures. Expected effects of sedation include bradycardia, hypotension, and hypoventilation. Oxygen therapy was continued throughout the procedure to maximize oxygen saturation levels. The following individualized sedatives were used, either alone or in combination: A: Dexmedetomidine 0.5 mg / m2 , reversal (atipamazole 0.05-0.2 mg / lb) B: Opioid-butorphanol (0.05-0.1 mg / lb), C: Performed with propofol-iv D: Benzodiazepine (diazepam): 0.1-0.2 mg / lb Subjects remained in the hospital facility for the day (<12 hours) and were then allowed to return home.

[0081] Clinical evaluations included owner's Canine Brief Pain Inventory behavioral assessment, Veterinary Clinical Visual Analog Scale for pain and mobility, bevel test, drug reduction / dependence, and video and gait monitoring. Figure 1 shows the results in bar graph form representing the clinical evaluation of functional improvement and pain relief achieved in the treatment of osteoarthritis of the forelimb joints in dogs after administration of the therapeutic IL-10 expression construct of the present invention. It is noted that administration of the IL-10 expression construct of the present invention resulted in significant functional improvement and pain relief in all of walking, trotting, palpation, range of motion, and functional impairment, especially at the 11 week time point.

[0082] Figure 2 shows the results in bar graph form representing owner assessment of improved function and reduced pain achieved in treating osteoarthritis of the forelimb joints in dogs following administration of a therapeutic IL-10 expression construct of the invention. Note again that there was a significant improvement at one week in all parameters of activity, quality of life, standing, walking, running and stair climbing.

[0083] 3 shows the results in bar graph form (pooled data) illustrating the clinical assessment of the functional improvement and pain relief achieved in the treatment of osteoarthritis of the forelimb joints in dogs following administration of a therapeutic IL-10 expression construct of the invention. The results of the clinical assessment show significantly positive results, especially in pain.

[0084] Figure 4 shows results in bar graph form (pooled data) depicting owner assessment of improved function and reduced pain achieved in the treatment of osteoarthritis of the forelimb joints in dogs following administration of a therapeutic IL-10 expression construct of the invention. Note that in these results, similar to the results shown in Figure 2, there was a significant improvement in all parameters, activity, quality of life, standing, walking, running and stair climbing, even at the one week time point.

[0085] 5 shows results (pooled data) demonstrating the improvement in range of motion achieved in treating osteoarthritis of the forelimb joints in dogs following administration of a therapeutic IL-10 expression construct of the invention. Note that the change in angle shows a significant improvement.

[0086] The foregoing description of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. The description of the invention includes one or more embodiments and descriptions of certain modifications and alterations, but other modifications and alterations are within the scope of the invention, for example, as may be within the skill and knowledge of one of ordinary skill in the art after appreciating the present disclosure. The right to include alternative embodiments to the extent permitted, such as alternative, substitute and / or equivalent structures, functions, ranges or steps to those set forth in the claims, whether or not such alternative, substitute and / or equivalent structures, functions, ranges or steps are disclosed herein, is intended to be obtained, and no patentable subject matter is to be made available to the public. (Additional Note) The technical ideas that can be understood from the above-described embodiment and modified examples will be described. [Item 1] 1. A method of treating an inflammatory joint disease in a subject, comprising: injecting into the inflamed joint a therapeutic anti-inflammatory composition comprising a therapeutically effective amount of a bacterial or viral IL-10 expression construct; The method, wherein the IL-10 expression construct comprises a bacterial or viral backbone and a nucleic acid sequence encoding interleukin-10. [Item 2] 2. The method of claim 1, wherein the IL-10 expression construct is administered together with an adjuvant. [Item 3] 3. The method according to item 2, wherein the adjuvant is selected from D-mannose, sucrose, glucose, calcium phosphate, a dendrimer, an oligonucleotide, high molecular weight hyaluronic acid, or a liposome. [Item 4] 2. The method of claim 1, wherein the nucleic acid sequence encoding interleukin-10 has an amino acid substitution of the wild-type phenylalanine at amino acid position 129. [Item 5] 5. The method of claim 4, wherein the amino acid substitution is selected from the group consisting of serine, alanine, threonine, or cysteine. [Item 6] The nucleic acid sequence encoding interleukin 10 is F129S The method according to item 5, wherein the encoding is [Item 7] 2. The method of claim 1, wherein the plasmid DNA comprises at least one nuclear targeting sequence 5' to the IL-10 coding sequence. [Item 8] 2. The method of claim 1, wherein the plasmid DNA comprises at least one nuclear targeting sequence 3' to the IL-10 coding sequence. [Item 9] 2. The method of claim 1, further comprising a diluent. [Item 10] 2. The method of claim 1, wherein the joint is a knee, elbow, wrist, ankle, hip, shoulder, or spine. [Item 11] 2. The method according to item 1, wherein the inflammatory joint disease is arthritis, tendonitis, bursitis, ligament inflammation, synovitis, gout, and systemic lupus erythematosus. [Item 12] 1. A method of treating an inflammatory joint disease in a subject, comprising: injecting into the inflamed joint a therapeutic anti-inflammatory composition comprising a therapeutically effective amount of a bacterial or viral IL-10 expression construct; The method, wherein the IL-10 expression construct comprises a bacterial or viral backbone, a nucleic acid sequence encoding interleukin-10; and a microparticle encapsulating the expression construct. [Item 13] 13. The method of claim 12, wherein the nucleic acid sequence encoding interleukin-10 has an amino acid substitution of the wild-type phenylalanine at amino acid position 129. [Item 14] 14. The method of claim 13, wherein the amino acid substitution is selected from the group consisting of serine, alanine, threonine, or cysteine. [Item 15] The nucleic acid sequence encoding interleukin-10 is F129S 15. The method according to item 14, wherein the encoding is [Item 16] 11. The method of claim 10, wherein the polymer comprises poly(lactic acid-co-glycolic acid). [Item 17] 17. The method of claim 16, wherein the polymer comprises a 50:50 lactic acid-glycolic acid copolymer. [Item 18] 11. The method of claim 10, wherein the plasmid DNA comprises at least one nuclear targeting sequence 5' to the IL-10 coding sequence. [Item 19] 11. The method of claim 10, further comprising a diluent. [Item 20] 11. The method of claim 10, wherein the joint is a knee, elbow, wrist, ankle, hip, shoulder, or spine. [Item 21] 11. The method according to item 10, wherein the inflammatory joint disease is arthritis, tendonitis, bursitis, ligament inflammation, synovitis, gout, and systemic lupus erythematosus. [Item 22] 22. The method of claim 21, wherein the inflammatory joint disease is osteoarthritis. [Item 23] 1. A method of treating an inflammatory joint disease in a subject, comprising: injecting into the inflamed joint a therapeutic anti-inflammatory composition comprising 1-500 μg of a bacterial or viral IL-10 expression construct; The method, wherein the IL-10 expression construct comprises a bacterial or viral backbone, a nucleic acid sequence encoding interleukin-10; and 5-1000 μg of D-mannose. [Item 24] 24. The method of claim 23, wherein D-mannose is administered together with the IL-10 expression construct. [Item 25] 24. The method of claim 23, wherein D-mannose is administered up to 10 days prior to administration of the IL-10 expression construct. [Item 26] 25. The method of claim 24, wherein the nucleic acid sequence encoding interleukin-10 has an amino acid substitution of the wild-type phenylalanine at amino acid position 129. [Item 27] 27. The method of claim 26, wherein the amino acid substitution is selected from the group consisting of serine, alanine, threonine, or cysteine. [Item 28] The nucleic acid sequence encoding interleukin-10 is F129S 28. The method according to item 27, wherein the encoding is [Item 29] 25. The method of claim 24, wherein the plasmid DNA comprises at least one nuclear targeting sequence 5' to the IL-10 coding sequence. [Item 30] 25. The method of claim 24, wherein the plasmid DNA comprises at least one nuclear targeting sequence 3' to the IL-10 coding sequence. [Item 31] 25. The method of claim 24, wherein the plasmid DNA comprises at least one nuclear targeting sequence both 5' and 3' to the IL-10 coding sequence. [Item 32] 1. A method of treating an inflammatory joint disease in a subject, comprising: injecting into the inflamed joint a therapeutic anti-inflammatory composition comprising 1-500 μg of a bacterial or viral IL-10 expression construct; The IL-10 expression construct comprises a bacterial or viral backbone and an IL-10 F129S and at least one nuclear targeting sequence either 5' or 3' to the IL-10 coding sequence; and 5 to 1000 μg of D-mannose.

Claims

[Claim 1] The invention described herein.

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