(19)
(11)EP 3 021 873 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
05.09.2018 Bulletin 2018/36

(21)Application number: 14826787.5

(22)Date of filing:  17.07.2014
(51)Int. Cl.: 
A61K 48/00  (2006.01)
(86)International application number:
PCT/US2014/047071
(87)International publication number:
WO 2015/009955 (22.01.2015 Gazette  2015/03)

(54)

COMPOSITION FOR THE TREATMENT OF INFLAMMATORY JOINT DISEASE

ZUSAMMENSETZUNG ZUR BEHANDLUNG VON ENTZÜNDLICHEN GELENKERKRANKUNGEN

COMPOSITION PUR LE TRAITEMENT D'UNE MALADIE INFLAMMATOIRE ARTICULAIRE


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 18.07.2013 US 201361847851 P

(43)Date of publication of application:
25.05.2016 Bulletin 2016/21

(60)Divisional application:
18167083.7 / 3369435

(73)Proprietor: Xalud Therapeutics, Inc.
San Francisco, CA 94110 (US)

(72)Inventors:
  • CHAVEZ, Raymond, A.
    San Francisco, CA 94110 (US)
  • WATKINS, Linda, R.
    Boulder, CO 80309 (US)
  • LANDRY, Robert
    Lafayette, CA 80026 (US)

(74)Representative: Green, Katherine et al
Mewburn Ellis LLP City Tower 40 Basinghall Street
London EC2V 5DE
London EC2V 5DE (GB)


(56)References cited: : 
WO-A1-2013/103966
US-A1- 2005 203 032
US-A1- 2009 035 256
US-B2- 7 846 428
WO-A2-2006/130580
US-A1- 2006 116 321
US-A1- 2013 172 410
  
  • WHALEN ET AL: "Adenoviral transfer of the viral IL-10 gene periarticularly to mouse paws suppresses development of collagen-induced arthritis in both injected and uninjected paws", THE JOURNAL OF IMMUNOLOGY, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, US, vol. 162, no. 6, 15 March 1999 (1999-03-15), pages 3625-3632, XP002149335, ISSN: 0022-1767
  • DEBORAH J GORTH ET AL: "IL-1ra delivered from poly(lactic-co-glycolic acid) microspheres attenuates IL-1[beta]-mediated degradation of nucleus pulposus in v", ARTHRITIS RESEARCH AND THERAPY, BIOMED CENTRAL, LONDON, GB, vol. 14, no. 4, 3 August 2012 (2012-08-03) , page R179, XP021117544, ISSN: 1478-6354, DOI: 10.1186/AR3932
  • SLOANE ET AL.: 'Immunological priming potentiates non-viral anti-inflammatory gene therapy treatment of neuropathic pain: Harnessing innate immunity to enhance gene therapy' GENE THERAPY vol. 16, no. 10, 02 July 2009, pages 1210 - 1222, XP055308809
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description


[0001] This application claims the benefit of US Provisional Patent Application No. 61/847,851, filed July 18, 2013.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH



[0002] This invention was made with government support under grant number U44-NS071642 awarded by the National Institute of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION



[0003] This invention relates compositions for treating clinical conditions associated with inflammatory diseases of the joints and symptoms associated therewith by administering to the inflamed joint the composition comprising a bacterial plasmid interleukin-10 (IL-10) expression construct.

BACKGROUND OF THE INVENTION



[0004] In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an "admission" of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under applicable statutory provisions.

[0005] Joint pain can flare up for any number of reasons, as a result of, e.g., over a hundred different arthritic conditions-of which rheumatoid arthritis and osteoarthritis are the most common-as well as tendonitis, bursitis, inflammation of the ligament, 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. Next, anti-inflammatory compounds take over to heal the area once the threat is diminished. When this process-known as local or acute inflammation takes place-it is a sign of a healthy immune system. However, if inflammation persists it can lead to a more chronic condition.

[0006] Current treatments for inflammatory diseases of the joint such as rheumatoid and osteo-arthritis, tendonitis, synovitis and the like remain suboptimal. Identifying treatments that would require less frequent administration would impact significantly the quality of life for patients with inflammatory joint disease; however, despite substantial research into and development of therapies for such conditions there is still a large unmet need for safe, effective, and easy-to-administer treatments.

SUMMARY OF THE INVENTION



[0007] The present invention provides a composition comprising a bacterial plasmid IL-10 expression construct, wherein the IL-10 expression construct comprises a bacterial backbone and a nucleic acid sequence encoding interleukin-10 for use in a method of treating inflammatory joint disease in a subject, said method comprising injecting the composition into an inflamed joint of the subject. In some embodiments, the IL-10 expression construct is encapsulated in biodegradable microparticles, and in certain aspects of these embodiments, the microparticles are suspended in a diluent to form a therapeutic composition.

[0008] In yet other, alternative embodiments, the therapeutic compositions of the present invention are IL-10 expression constructs-with a bacterial backbone-that are delivered as "naked" DNA. That is, the IL-10 expression constructs are delivered without encapsulation. In such embodiments, the IL-10 expression constructs are delivered in conjunction with one or more diluents, and in preferred aspects of these embodiments, the IL-10 expression construct is delivered in conjunction with one or more adjuvants. In some aspects of this embodiment, the one or more adjuvants may be administered to a subject at the time of the administration of the IL-10 expression construct or up to 10 days before administration of the IL-10 expression construct, e.g., as a "pretreatment".

[0009] In some aspects, the joint inflammation is in the knee, elbow, wrist, ankle, hip, shoulder, or spine. Conditions treatable by the methods of the present invention include rheumatoid arthritis and osteoarthritis, as well as tendonitis, bursitis, inflammation of the ligament, synovitis, gout, and systemic lupus erythematosus.

[0010] Thus, in some embodiments the composition comprises: plasmid DNA comprising a bacterial backbone and at least one IL-10 coding sequence and a diluent; where the therapeutic anti-inflammatory IL-10 compound provides a therapeutically effective dose to a joint at about 1 µg DNA to about 1000 µg DNA, or about 5 µg DNA to about 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, though preferably about 2.5 µg DNA to about 500 µg DNA. In other cases, a viral vector is used as an alternative to a bacterial vector.

[0011] In some preferred embodiments of the present invention, the IL-10 therapeutic compounds of the present invention (i.e., encapsulated or naked DNA) are administered with one or more adjuvants. The adjuvants of the invention are biocompatible agents that may be administered simultaneously with the IL-10 therapeutic compounds or as a pretreatment before the IL-10 therapeutic compounds are administered. Adjuvants that are particularly preferred include but are not limited to mannose, sucrose, glucose, calcium phosphate, dendrimers, liposomes including cationic liposomes, and oligodeoxynucleotides. The adjuvants of the present invention in preferred embodiments are those adjuvants that increase uptake or efficacy of the IL-10 expression construct. Concurrent administration or pretreatment includes administering to the joint about 5 µg adjuvant to about 1000 µg adjuvant, or about 10 µg adjuvant to about 750 µg adjuvant, or about 50 µg adjuvant to about 500 µg adjuvant, or about 25 µg adjuvant to about 750 µg adjuvant at the time of or up to 10 days prior to the administration of the therapeutic anti-inflammatory IL-10 compound.

[0012] Other alternative embodiments provide compositions for use in methods for treatment of inflammatory diseases of the joints wherein the composition comprises: an IL-10 expression construct; microparticles encapsulating the IL-10 expression construct; and a diluent; where the therapeutic microparticle composition provides a therapeutically effective dose to a joint at 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 or about 25 µg DNA to about 100 µg DNA.

[0013] In yet other embodiments are methods for slowing progression of inflammatory diseases of the joints in a subject 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, where the therapeutic IL-10 expression construct provides a therapeutically effective dose to a joint at 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. In such embodiments, an adjuvant preferably is administered with the therapeutic anti-inflammatory IL-10 compound or up to 10 days before administration of the therapeutic anti-inflammatory IL-10 compound, e.g., as a pretreatment.

[0014] Optionally in the embodiments described, the IL-10 expression construct comprises at least one nuclear targeting sequence located either from 100 to 2000 bp 5' to the at least one IL-10 coding sequence and/or from 150 to 450 bp 3' to the at least one IL-10 coding sequence. In other aspects of the present invention, the IL-10 expression construct comprises two nuclear targeting sequences where one nuclear targeting sequence is positioned from 100 to 2000 bp 5' of the at least one IL-10 coding sequence, and one nuclear targeting sequence is positioned from 150 to 450 bp 3' of the at least one coding sequence.

[0015] In the invention, the therapeutic composition is administered by injection into the joint(s), and in preferred embodiments the therapeutic composition is administered by intra-articular injection.

[0016] In some aspects, the nucleic acid sequence encoding interleukin-10 has an amino acid substitution for wildtype 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. In some aspects, the nucleic acid sequence encoding interleukin-10 encodes IL-10F129S. In yet other aspects, the IL-10 expression construct comprises at least one nuclear targeting sequence 5' to the IL-10 coding sequence, and in some aspects, IL-10 expression construct comprises at least one nuclear targeting sequence 3' to the IL-10 coding sequence.

[0017] In some aspects of the present invention, the microparticles comprise one or more of poly(2-hydroxy ethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid), polylactides (PLA), polyglycolides (PGA), poly(lactide-co-glycolides) (PLGA), polyanhydrides, polycaprolactone, poly-3-hydroxybutyrate or polyorthoesters. In certain aspects of the present invention, the microparticles comprise PLGA. In other aspects of the invention, the microparticles comprise a mixture of biodegradable polymers representing different, complimentary release profiles.

[0018] In certain embodiments of the methods, the therapeutic anti-inflammatory IL-10 expression construct (and any adjuvant or "pretreatment" adjuvant), is delivered approximately every 40 to 120 days as needed for therapeutic effect, e.g., up to one year. In other embodiments, the therapeutic anti-inflammatory composition is delivered approximately every 40 to 120 days as needed for therapeutic effect for greater than one year. In yet other embodiments, the therapeutic anti-inflammatory composition is delivered as needed for therapeutic effect approximately every 40 to 120 days, as needed, for the life of the subject.

[0019] The methods described herein also may be employed as a research tool to identify pharmaceuticals, small molecules and/or biologics that may be used in conjunction in a "cocktail" with the therapeutic IL-10 expression construct compositions.

DESCRIPTION OF THE FIGURES



[0020] 

Figure 1 shows results in the form of bar graphs illustrating clinical assessment of the functional improvement and pain reduction achieved treating osteoarthritis of forelimb joints in canines after administration of the therapeutic IL-10 expression constructs of the present invention.

Figure 2 shows results in the form of bar graphs illustrating owner assessment of the functional improvement and pain reduction achieved treating osteoarthritis of forelimb joints in canines after administration of the therapeutic IL-10 expression constructs of the present invention.

Figure 3 shows results in the form of bar graphs illustrating clinical assessment of the functional improvement and pain reduction achieved treating osteoarthritis of forelimb joints in canines after administration of the therapeutic IL-10 expression constructs of the present invention (pooled data).

Figure 4 shows results in the form of bar graphs illustrating owner assessment of the functional improvement and pain reduction achieved treating osteoarthritis of forelimb joints in canines after administration of the therapeutic IL-10 expression constructs of the present invention (pooled data).

Figure 5 shows results illustrating improvements in range of motion achieved treating osteoarthritis of forelimb joints in canines after administration of the therapeutic IL-10 expression constructs of the present invention (pooled data).


DETAILED DESCRIPTION OF THE INVENTION


Definitions



[0021] Unless defined otherwise, 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 for the purpose of describing and disclosing devices, animal models, formulations and methodologies that may be used in connection with the presently described invention.

[0022] The term adjuvant as used herein refers to a pharmacological or immunological agent that modifies the effect of other agents. In the context of the present invention, an adjuvant is used to increase the efficacy of the therapeutic IL-10 anti-inflammatory compound. Adjuvants of particular utility in the present invention include those that enhance the uptake or efficacy of the IL-10 expression construct by, e.g., macrophages or other immune cells present in the synovial fluid of the joint.

[0023] The term "anti-inflammatory" as used herein refers to decreasing the action or production of one or more proinflammatory cytokines or proteins produced by nerves, neurons, glial cells, endothelial cells, fibroblasts, muscle, immune cells or other cell types.

[0024] The term "anti-inflammatory cytokine" as used herein refers to a protein that decreases the action or production of one or more proinflammatory cytokines or proteins produced by nerves, neurons, glial cells, endothelial cells, fibroblasts, muscle, immune cells or other cell types. Inflammatory cytokines and proteins include, without limitation, interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-a), interleukin-6 (IL-6), inducible nitric oxide synthetase (iNOS) and the like. Non-limiting examples of anti-inflammatory cytokines include interleukin-10 (IL-10) including viral IL-10, interleukin-4 (IL-4), interleukin-13 (IL-13), alpha-MSH, transforming growth factor-beta 1 (TGFβ1), and the like. Thus, the full-length molecules and fragments of anti-inflammatory cytokines, as well as anti-inflammatory cytokines with modifications, such as deletions, additions and substitutions (either conservative or non-conservative in nature), to the native sequence, are intended for use herein, so long as the anti-inflammatory cytokine is therapeutically effective. Modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. Accordingly, active proteins are typically substantially homologous to the parent sequence, e.g., proteins are typically about 70 ... 80 ... 85 ... 90 ... 95 ... 98. .. 99%, etc. homologous to the parent sequence.

[0025] A "coding sequence" of an anti-inflammatory cytokine or 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 control sequences. The boundaries of the coding sequence are determined by nucleotides corresponding to a start codon at the amino terminus and nucleotides corresponding to a translation stop codon at the carboxy terminus.

[0026] The term DNA "control sequences" refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, enhancers, and the like, which collectively provide for 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.

[0027] The terms "effective amount" or "therapeutically effective amount" of a therapeutic IL-10 expression construct used in the methods of the invention refer to a nontoxic but sufficient amount of the IL-10 expression construct to provide the desired response, such as a decrease in inflammation of the joints, relief from symptoms caused by inflammatory diseases of the joints and/or preventing progression of joint damage due to inflammatory diseases of the joints. The exact amount required will 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 particular IL-10 expression construct to be delivered whether the IL-10 expression construct is delivered in a microparticle or as naked DNA, mode of administration (e.g., intra-articular injection), and the like. Dosage parameters for the present methods are provided herein; however, optimization of an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using the methods set forth herein and routine experimentation.

[0028] The term "excipient" or "diluent" refers to an inert substance added to a therapeutic composition of the invention to facilitate administration of the therapeutic IL-10 expression construct. Examples, without limitation, of excipients include saline, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, hyaluronic acid optionally formulated with a surfactant, Plurnoic F-68, vegetable oils and polyethylene glycols.

[0029] By "isolated" when referring to a nucleotide sequence, is meant that the expression construct is present in the substantial absence of other biological macromolecules of the same type. Thus, an "isolated nucleic acid molecule which encodes a particular polypeptide" refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties that do not deleteriously affect the basic characteristics of the composition.

[0030] The term "joint" refers to an 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, bursae and synovium. Joints that can be treated with the methods herein include fixed, hinge, pivot or ball-and-socket joints.

[0031] The term "joint inflammation" refers to all types of arthritis caused by inflammation where rheumatoid arthritis, osteoarthritis are the most common, as well as tendonitis, bursitis, inflammation of the ligament, synovitis, gout, and systemic lupus erythematosus.

[0032] The term "nuclear targeting sequence" refers to a nucleic acid sequence which functions to improve the expression efficiency of an anti-inflammatory cytokine in a cell.

[0033] "Operably linked" refers to an arrangement of elements where the components so described are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control sequences need not be contiguous with the coding sequence so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0034] The term "promoter" is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters".

[0035] For the purpose of describing the relative position of nucleotide sequences in a particular nucleic acid molecule throughout the instant application, such as when a particular nucleotide sequence is described as being situated "upstream," "downstream," "3 prime (3')" or "5 prime (5')" relative to another sequence, it is to be understood that it is the position of the sequences in the "sense" or "coding" strand of a DNA molecule that is being referred to as is conventional in the art.

[0036] The term "research tool" as used herein refers to any methods of the invention using the therapeutic IL-10 expression constructs for scientific inquiry, either academic or commercial in 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 subject to regulatory approval; rather, the research tools of the invention are intended to facilitate research and aid in such development activities, including any activities performed with the intention to produce information to support a regulatory submission.

[0037] The terms "subject", "individual" or "patient" may be used interchangeably herein and refer to a vertebrate, preferably a mammal.

[0038] The term "therapeutic composition" or "therapeutic anti-inflammatory composition" as used herein refers to a composition that has the ability to decrease inflammation of the joints, provide relief from symptoms caused by inflammatory diseases of the joints and/or prevent progression of joint damage due to inflammatory diseases of the joints as measured in any of the known animal models or by assessment performed in humans.

[0039] "Treatment" or "treating" joint inflammation includes: (1) decreasing inflammation of the joint or causing the inflammation to occur with less intensity in a subject that may be predisposed to joint inflammation but does not yet experience or display symptoms, or (2) inhibiting joint inflammation, i.e., arresting the development of or reversing symptoms or physiological damage caused by inflammation.

[0040] A "viral vector" as used herein is a recombinantly produced virus or viral particle that comprises an IL-10 expression construct to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.

[0041] 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 techniques), cell biology, biochemistry, and sequencing technology, which are within the skill of those who practice in the art. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found 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 (W.H. 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, Fourth Ed. (W.H. Freeman); Gait (1984), "Oligonucleotide Synthesis: A Practical Approach" (IRL Press); Nelson and Cox (2000), Lehninger, Principles of Biochemistry, Third Ed. (W. H. Freeman); and Berg et al. (2002) Biochemistry, Fifth Ed. (W.H. Freeman);.

[0042] Note that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0043] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0044] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific 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 invention.

Methods of the Invention



[0045] The present invention provides compositions for use in methods for treating inflammatory diseases of the joints, symptoms associated with inflammatory diseases of the joints, and slowing disease progression by administering to a subject-typically injected via intra-articular 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. As an alternative, in preferred embodiments the therapeutic IL-10 expression vector is delivered as a "naked" vector, where the delivery of the naked IL-10 expression vector is accompanied by or preceded by administration of a nucleic acid uptake adjuvant. The nucleic acid uptake adjuvant may be administered either concurrently or up to ten or more days prior to the administration of the naked IL-10 expression vector. The methods of the present invention may be used to treat joint inflammation in the knee, elbow, wrist, ankle, hip, shoulder, or spine. Conditions treatable by the methods of the present invention include rheumatoid arthritis and osteoarthritis, as well as tendonitis, bursitis, inflammation of the ligament, synovitis, gout, and systemic lupus erythematosus.

[0046] Thus, the invention generally provides the compositions for use in methods for treating inflammatory diseases of the joints and symptoms and physiological damage associated with inflammatory diseases of the joints. The disclosure also provides for using the methods in research of inflammatory diseases of the joints, including identifying pharmaceuticals, small molecules and/or biologics that may be used in conjunction (in a "cocktail") with the therapeutic IL-10 expression construct. The methods comprise the step of administering to a subject, preferably by injection into a joint, an IL-10 expression construct comprising a bacterial or viral backbone and at least one IL-10 coding sequence. In some cases the IL-10 expression construct is optionally encapsulated in biodegradable microparticles. The anti-inflammatory compositions are generally suspended in a diluent for delivery to a joint. The IL-10 expression construct may comprise at least one nuclear targeting sequence where the at least one nuclear targeting sequence is 5', 3' or both to the IL-10 coding sequence.

[0047] The anti-inflammatory compounds may consist of a "naked" bacterial vector or viral vector capable of expressing the at least one IL-10 coding sequence, or the anti-inflammatory compound may consist of a bacterial or viral vector encased in microparticles or other delivery device. Administration of the therapeutic IL-10 expression construct may be accompanied by or preceded by administration of an adjuvant, and in the case of delivery of naked IL-10 expression constructs, a nucleic acid uptake adjuvant may be administered concurrently or up to ten days or more before administration of the naked IL-10 expression construct.

[0048] The IL-10 expression construct for the use in the method of treatment of the present invention comprises a bacterial backbone (plasmid DNA or pDNA) 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 control sequences. Optionally, the pDNA also may comprise one or more additional anti-inflammatory cytokine coding sequences, and/or a marker sequence to allow for selection of transformed cells during amplification of the pDNA. The bacterial backbone can be any bacterial backbone known to those with skill in the art. Backbones typically selected are those that, e.g., contain or lack appropriate restriction sites to allow ease of cloning, may be produced and isolated with ease, are not immunogenic, and the like. For example, bacterial backbones derived from E. coli are of use in the present invention.

[0049] The plasmid DNA comprises at least one IL-10 coding sequence. IL-10 coding sequence may code for wildtype IL-10, or the IL-10 may be a mutant IL-10. One mutant IL-10 of interest contains one or more mutations that cause amino acid substitutions, additions or deletions as compared to wildtype IL-10 in the "hinge" region of the IL-10 protein. The human IL-10 protein is a homodimer, where each monomer comprises six alpha helices A→F, the length of which are 21, 8, 19, 20, 12 and 23 amino acids, respectively. Helices A→D of one monomer noncovalently interact with helices E and F of a second monomer, forming a noncovalent V-shaped homodimer. The "hinge" region targeted for mutation according to the present invention comprises the amino acids between the D and E alpha helices on one or both monomers at approximately amino acid position X to position Y of wildtype IL-10 . For example, mutant rat and human IL-10 proteins have been described in which the phenylalanine at position 129 of the wildtype sequence has been replaced with a serine residue. (See, e.g., Sommer, et al., WO2006/130580 and Milligan, et al., Pain, 126:294-308 (2006).) The resulting mutant IL-10 is referred to as IL-10F129S. Other substitutions for the wildtype phenylalanine at amino acid position 129 may be, e.g., threonine, alanine, or cysteine. Thus the present invention in yet another aspect encompasses one or more substitutions at amino acid position 129 or at other amino acids within the hinge region of the IL-10 protein, or functional equivalents thereof.

[0050] Additional anti-inflammatory cytokines of use in the present invention include but are not limited to interleukin-4 (IL-4), interleukin-13 (IL-13), alpha-MSH, transforming growth factor-beta 1 (TGFβ1) and the like.

[0051] Nuclear targeting sequences of the present invention are sequences that promote expression of the protein(s) encoded by the at least one IL-10 coding sequence and the optional, additional anti-inflammatory cytokine coding sequence(s). For example, in one aspect the nuclear targeting sequences may bind to nuclear transport chaperone proteins, facilitating uptake of the plasmid DNA by the cell nucleus. Such sequences include but are not limited to interspersed (or dispersed) DNA repeats or repetitive sequences such as transposable elements, flanking or terminal repeats such as the long terminal repeats (LTRs) on retrovirus genomes such as SV40s, tandem repeats, and the inverted terminal repeats (ITRs) of viral genomes such as Adeno-Associated Virus and Adenovirus. In other aspects, the nuclear targeting sequences are sequences that act to bind transcription factors for import into the nucleus, such as enhancer sequences.

[0052] In addition to a bacterial backbone, at least one IL-10 coding sequence and optional one or more additional anti-inflammatory cytokine coding sequences and, optionally, one or more nuclear targeting sequences, the plasmid DNA comprises one or more DNA control sequences, such as promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites and the like, which collectively provide for the replication, transcription and translation of the anti-inflammatory cytokine coding sequence(s) in a recipient cell. Not all of these control sequences need always be present so long as the anti-inflammatory cytokine coding sequences are capable of being replicated, transcribed and translated in an appropriate host cell. Promoter sequences of use herein include but are not limited to chicken or human β-actin promoters, cytomegalovirus immediate early promoters, glyceraldehydes 3-phosphate dehydrogenase (GADPH) promoters, elongation factor 1α (eF1α) promoter, GFAP promoter, murine leukemia virus (MLV) promoter, herpes simples virus thymidine kinase (TK) promoter, and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) promoters; upstream regulatory domains of use in the present invention include but are not limited to cytomegalovirus immediate early promoter enhancers, mouse mammary tumor virus (MMTV) enhancer and simian virus 40 (SV40) enhancer; and 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 will also comprise a selection marker gene, such as that coding for antibiotic resistance. Marker genes of use herein include but are not limited to neomycin, hygromycin-B, ampicillin, kanomycin, or puromycin.

[0053] For example, plasmids comprising the 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 were used in some experiments demonstrating the usefulness of the present invention. In other experiments, plasmids comprising the 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 were used. Details of these plasmids are disclosed in Milligan, et al., Pain 126:294-308 (2006).

[0054] 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 categorized into two groups according to whether their genomes integrate into host cellular chromatin (oncoretroviruses and lentiviruses) or persist in the cell nucleus predominantly as extrachromosomal episomes (adeno-associated virus, adenoviruses, herpesviruses, and integration-deficient lentiviruses). For example, in one embodiment of the present invention, viruses from the Parvoviridae family are utilized. The Parvoviridae is a family of small single-stranded, non-enveloped DNA viruses with genomes approximately 5000 nucleotides long. Included among the family members is adeno-associated virus (AAV), a dependent parvovirus that by definition requires co-infection with another virus (typically an adenovirus or herpesvirus) to initiate and sustain a productive infectious cycle. In the absence of such a helper virus, AAV is still competent to infect or transduce a target cell by receptor-mediated binding and internalization, penetrating the nucleus in both non-dividing and dividing cells.

[0055] Another viral delivery system useful with the IL-10 expression constructs of the present invention is a system based on viruses from the family Retroviridae. Retroviruses comprise single-stranded RNA animal viruses that are characterized by two unique features. First, the genome of a retrovirus is diploid, consisting of two copies of the RNA. Second, this RNA is transcribed by the virion-associated enzyme reverse transcriptase into double-stranded DNA. This double-stranded DNA or provirus can then integrate into the host genome and-be passed from parent cell to progeny cells as a stably-integrated component of the host genome.

[0056] In some cases, lentiviruses are the preferred members of the retrovirus family for use herein. Lentivirus vectors are often pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G), and have been derived from the human immunodeficiency virus (HIV), the etiologic agent of the human acquired immunodeficiency 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 immune deficiency in cats; bovine immunodeficiency virus (BIV) which causes lymphadenopathy and lymphocytosis in cattle; and simian immunodeficiency virus (SIV), which causes immune deficiency and encephalopathy in non-human primates. Vectors that are based on HIV generally retain <5% of the parental genome, and <25% of the genome is incorporated into packaging constructs, which minimizes the possibility of the generation of reverting replication-competent HIV. Biosafety has been further increased by the development of self-inactivating vectors that contain deletions of the regulatory elements in the downstream long-terminal-repeat sequence, eliminating transcription of the packaging signal that is required for vector mobilization. The main advantage to the use of lentiviral vectors is that gene transfer is persistent in most tissues or cell types due to integration of the viral vector. However, lentivirus that is integrase deficient could also be used.

[0057] Adenoviruses (Ads) are a relatively well characterized homogenous group of viruses, including over 50 serotypes. See, e.g., International PCT Application No. WO 95/27071. Adenoviruses are medium-sized (90-100 nm), nonenveloped (without an outer lipid bilayer) icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. There are 57 described serotypes in humans, which are responsible for 5-10% of upper respiratory infections in children, and many infections in adults as well. They are classified as group I under the Baltimore classification scheme, meaning their genomes consist of double-stranded DNA, and are the largest nonenveloped viruses. Because of their large size, they are able to be transported through the endosome (i.e., envelope fusion is not necessary). The virion also has a unique "spike" or fiber associated with each penton base of the capsid that aids in attachment to the host cell via the coxsackie-adenovirus receptor on the surface of the host cell.

[0058] The adenovirus genome is linear, non-segmented double-stranded (ds) DNA that is between 26 and 45 kb, allowing the virus to theoretically carry 22 to 40 genes. Although this is significantly larger than other viruses in its Baltimore group, it is still a very simple virus and is heavily reliant on the host cell for survival and replication. Once the virus has successfully gained entry into the host cell, the endosome acidifies, which alters virus topology by causing capsid components to disassociate. With the help of cellular microtubules, the virus is transported to the nuclear pore complex, where the adenovirus particle disassembles. Viral DNA is subsequently released, which can enter the nucleus via the nuclear pore. After this the DNA associates with histone molecules. Thus, viral gene expression can occur and new virus particles can be generated.

[0059] Unlike most lentiviral vectors (i.e., those that can integrate, e.g., are not integrase deficient), adenoviral DNA does not integrate into the genome and is not replicated during cell division. The primary applications for adenovirus are in gene therapy and vaccination. Recombinant adenovirus-derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed. See, International PCT Application Nos. WO 95/00655 and WO 95/11984.

[0060] Other viral or non-viral systems known to those skilled in the art also may be used to deliver IL-10 expression constructs to the joint, including but not limited to gene-deleted adenovirus-transposon vectors that stably maintain virus-encoded transgenes in vivo through integration into host cells (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 mini-circle DNA vectors devoid of bacterial DNA sequences (see Chen, et al., Molecular Therapy. 8(3):495-500 (2003)). Mini-circle DNA as described in U.S. Patent Publication No. 2004/0214329 discloses vectors that provide for persistently high levels of nucleic acid transcription.

[0061] Once the IL-10 expression construct has been constructed, amplified and isolated by techniques known in the art, the IL-10 expression construct is then optionally, in some embodiments, encapsulated within microparticles. Techniques for encapsulating IL-10 expression construct vary depending on the type of microparticles used and such techniques are described in more detail infra. The microparticles may be comprised of any biodegradable polymer. To be used successfully as a biodegradable polymer in the controlled drug delivery formulations, the material must be chemically inert and free of leachable impurities. Ideally the polymer also has an appropriate physical structure, with minimal undesired aging, and is readily processable. Some of the materials include poly(2-hydroxy ethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol), poly(acrylic acid), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), and poly(methacrylic acid). Biodegradable polymers of particular use include polylactides (PLA), polyglycolides (PGA), poly(lactide-co-glycolides) (PLGA), polyanhydrides, polycaprolactone, poly-3-hydroxybutyrate and polyorthoesters. Such biodegradable polymers have been characterized extensively and can be formulated to exhibit desired degradation properties as is known in the art (see, e.g., Edlund & Albertsson, Degradable Aliphatic Polyesters, pp. 67-112 (2002), Barman, et al., J. of Controlled Release, 69:337-344 (2000); Cohen, et al., Pharmaceutical Res., (8): 713-720 (1991)).

[0062] In one particular embodiment of the invention, the polymer comprises poly(lactide-co-glycolides) (PLGA). PLGA is a copolymer which is used in a host of FDA approved therapeutic devices, owing to its biodegradability and biocompatibility. PLGA is synthesized by means of random ring-opening co-polymerization of two different monomers, the cyclic dimers (1,4-dioxane-2,5-diones) of glycolic acid and lactic acid. Common catalysts used in the preparation of this polymer include tin(II) 2-ethylhexanoate, tin(II) alkoxides, or aluminum isopropoxide. During polymerization, successive monomeric units of glycolic or lactic acid are linked together in PLGA by ester linkages, thus yielding a linear, aliphatic polyester as a product.

[0063] Depending on the ratio of lactide to glycolide used for the polymerization, different forms of PLGA can be obtained: these are usually identified in regard to the monomers' ratio used (e.g., PLGA 75:25 identifies a copolymer whose composition is 75% (molar percent) lactic acid and 25% (molar percent) glycolic acid). PLGA degrades by hydrolysis of its ester linkages in the presence of water. It has been shown that the time required for degradation of PLGA is related to the monomers' ratio used in production: the higher the content of glycolide units, the lower the time required for degradation. An exception to this rule is the copolymer with 50:50 monomers' ratio which exhibits the faster degradation (about two months). In addition, polymers that are end-capped with esters (as opposed to the free carboxylic acid) demonstrate longer degradation half-lives. Of particular use in the present invention is PLGA having a composition of between 20% and 80% lactic acid and between 80% and 20% glycolic acid. More preferred for use herein is PLGA having a composition of between 65% and 35% lactic acid and between 35% and 65% glycolic acid. In one aspect, PLGA having a composition of 50% lactic acid and 50% glycolic acid is used.

[0064] Additionally, the IL-10 expression constructs (pDNA) may be encapsulated in batches of microparticles having different release profiles; for example, 10% of the pDNA to be delivered may be encapsulated in microparticles having, e.g., a one day to four week release profile; 30% of the pDNA to be delivered may be encapsulated in microparticles having, e.g., a three week to six week release profile; 30% of the pDNA to be delivered may be encapsulated in microparticles having, e.g., a six week to ten week release profile; and 30% of the pDNA to be delivered may be encapsulated in microparticles having, e.g., an eight week to twelve week release profile. In such an embodiment, a single type of biodegradable polymer may be used, but used in formulations with different release profiles; alternatively, different biodegradable polymers having different release characteristics may be used. In yet another embodiment, the formulation of the microparticles may be varied so as to change the surface of the microparticles to enhance or retard, as desired, the travel of the therapeutic composition through, e.g., the synovial fluid of the joint.

[0065] Once microparticles are obtained, they are suspended in an acceptable diluent to form a therapeutic composition for administration to a subject. Similarly, if the IL-10 expression constructs of the present invention are delivered as naked DNA as opposed to being encapsulated, diluents are also used for administration to the subject. Such diluents (or excipients) include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and that may be administered without undue toxicity. Pharmaceutically acceptable diluents may comprise sorbitol, alum, dextran, sulfate, large polymeric anions, any of the various TWEEN compounds, and liquids such as water, saline, glycerol or ethanol, oil and water emulsions, or adjuvants such as Freund's adjuvant. Pharmaceutically acceptable salts can be included therein as well, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like may be present in such vehicles. In one aspect of the invention, different diluents are used based on their ability to migrate through the targeted site in joint. For example, for delivery in an adult human, diluents may be preferred that favor more rapid spread of the therapeutic composition through the joint; conversely, in children or small animals where the size of the joint is less of a concern, a diluent may be used that does not disperse the therapeutic composition quickly. A thorough discussion of pharmaceutically acceptable diluents/excipients is available in Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins (2005). Preferred diluents include but are not limited to Physiosol®, artificial synovial fluid, preservative-free 0.9% NaCl, lactated Ringer's injection solution, and Elliotts B® Solution.

[0066] Synthesizing and administering the compositions for the inventive use involve a series of steps. First, a plasmid is constructed comprising the various components described supra. Then the plasmid DNA (pDNA) is amplified and isolated by techniques well known in the art. Once the pDNA is isolated, it may be combined with polymers to form pDNA-containing microparticles. Methods for microparticle formation vary depending on the polymers used, however, a double emulsion technique is typically employed. First, a polymer is dissolved in an organic solvent. Next, pDNA is suspended in an aqueous solution and is 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 by passage through a particulate medium producing turbulence, or the mixture can be sonicated. Most preferable is any method by which the nucleic acid receives the least amount of damage in the form of nicking, shearing, or degradation, while still allowing the formation of an appropriate emulsion. During this process, the polymer forms into microparticles, many of which contain pDNA. If desired, one can isolate a small amount of the nucleic acid at this point in order to assess integrity, e.g., by gel electrophoresis.

[0067] The first emulsion is then added to an organic solution. The solution can be comprised of, for example, methylene chloride, ethyl acetate, or acetone, typically containing polyvinyl alcohol (PVA), and often having approximately a 1:100 ratio of the weight of PVA to the volume of the solution. The first emulsion is generally added to the organic solution with stirring in a homogenizer or sonicator. This process forms a second emulsion which is subsequently added to another organic solution with stirring (e.g., in a homogenizer). In one aspect of this method, the latter solution is 0.05% w/v PVA. The resultant microparticles are washed several times with water to remove the organic compounds. In some aspects, more than approximately 40% of the resulting microparticles contain pDNA. In yet other aspects, more than approximately 50% of the resulting microparticles contain pDNA, in yet other aspects, more than 55% of the resultant microparticles contain pDNA.

[0068] The ability to internalize differently-sized microparticles varies with cell type. In certain embodiments, macrophages and antigen-presenting cells were targeted. Such cells more efficiently internalize microparticles of less than about 5 µ (see Shakweh, et al., Eur J of Pharmaceutics and Biopharmaceutics 61(1-2):1-13 (2005)). Thus, if desired, particles may be passed through sizing screens to selectively remove those larger than the desired size. In one particular aspect, microparticles of less than 5 µ are used in the therapeutic composition, and in other particular aspects, microparticles of less than 3 µ are used in the therapeutic composition. After washing, the particles can either be used immediately or be lyophilized for storage. The size distribution of the microparticles prepared by the methods described herein can be determined with, e.g, a Coulter™ counter or laser diffraction. Alternatively, the average size of the particles can be determined by visualization under a microscope fitted with a sizing slide or eyepiece. Alternatively, a scanning electron microscope can be used to assess both size and microparticle morphology.

[0069] Once IL-10 expression construct-containing microparticles are obtained, the microparticles can be suspended immediately in diluent or lyophilized for storage. The combination of the microparticles and diluent forms the therapeutic microparticle composition that can be administered by injection into a joint to an animal subject. The recombinant vectors can be introduced either in vivo or in vitro (also termed ex vivo) to treat joint inflammation. If transduced in vitro, the desired recipient cell or synovial fluid is removed from the subject, treated with pDNA-containing microparticles and reintroduced into the subject. Alternatively, syngeneic or xenogeneic cells can be transformed for delivery where such cells typically do not generate an inappropriate immune response in the subject. If administered in vivo, recombinant vectors or cells transformed with the vectors in vitro are delivered directly by injection into the joint.

[0070] The IL-10 expression constructs are, in an alternative embodiment, administered as naked DNA. In such a case, the IL-10 expression constructs are amplified, e.g., using good quality manufacturing practices. GMPs are enforced in the United States by the U.S. Food and Drug Administration (FDA), under Section 501(B) of the 1938 Food, Drug, and Cosmetic Act (21 USCS § 351).

[0071] Adjuvants appropriate for the present use include adjuvants that increase the uptake of the IL-10 expression constructs of the present invention; that is, adjuvants appropriate include any biologically-compatible agent that neutralizes or obviates the issue of introducing negatively-charged DNA into cells with a negatively-charged membrane. Such adjuvants include sugars such as mannose, glucose and sucrose; calcium phosphate; dendrimers (repetitively branched molecules); liposomes (spherical vesicles comprising a lipid bilayer) including cationic liposomes; DEAE-dextran including DEAE-dextran polyethylenimine; oligodeoxynucleotides; and high molecular weight hyaluronic acid (>1MDa), an anionic nonsulfated glycosaminoglycan.

[0072] One adjuvant of particular interest is D-mannose. D-mannose is a simple hexose sugar with a molecular weight of 180.2 and is known to: decrease inflammatory processes during wound healing (Kossi J, et al., Eur Surg Res, 31(1):74-82 (1999), reduce oxidative bursts required during inflammation (Rest RF, et al., J Leukoc Biol, 43(2):158-164 (1988)), suppress adjuvant-induced arthritis in a rat model (Willenborg DO, et al., Immunol Cell Biol, 70(Pt 6):369-377 (1996)), inhibit LPS-induced IL-1β, TNF-α, decrease NF-kB/p65 critical for proinflammatory cytokine expression, and decrease leukocyte influx following intratracheal instillation of LPS, which is a model of sepsis-associated acute lung injury and respiratory distress syndrome (Xu XL, et al., Inflamm Res, 57(3):104-110 (2008); Xu X, et al., Eur J Pharmacol, 641(2-3):229-237 2010)). The MR is a transmembrane glycoprotein pattern recognition receptor involved in host defense of innate immunity by recognizing mannosylated ligands (for example, lysosomal hydrolases) that can include a variety of bacteria, yeasts and parasites expressing 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, 479:1-14 (2000); Stahl PD, et al., Curr Opin Immunol, 10(1):50-55 (1998)).

[0073] Because the therapeutic compositions do not significantly induce an immune response or dose tolerance in subjects, they can be administered as needed for therapeutic effect. That is, the therapeutic anti-inflammatory composition can be delivered approximately every 40 to 120 days (or as required) as needed for therapeutic effect for shorter-term therapy. However, when longer-term therapy is desired, the therapeutic composition can be delivered approximately every 40 to 120 days (or more or less) as needed for therapeutic effect for greater than one year; and if necessary, for the life of the subject. Dosage frequency depends on the dosage, the adjuvant used and the health of the subject.

[0074] Dosage ranges of the therapeutic compositions used in the methods of the present invention vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, joint site, and the particular IL-10 expression construct to be delivered, whether or not the IL-10 expression construct if encapsulated, mode of administration, and the like. Dosage ranges include a therapeutically effective dose per joint at 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.

[0075] The IL-10 expression constructs or microparticles containing the IL-10 expression constructs used in the methods of the present invention may be co-administered in a "cocktail" with other therapeutic agents useful in treating joint inflammation including glucocorticoids; methotrexate; hydroxychlolquine; sulfasalazine; lefunomide; anti-TNF agents such as etanercept, infliximab and adalimumab; abatacept; hyaluronic acid, particularly high molecular weight hyaluronic acid (>1MDa) such as Hyalgan, Orthovisc, or Synvisc at a dose of, e.g., 0.5-2.5% (5 to 25 mg/mL) from 1 to 5 mL, so from 5 mg to 125 mg per joint; and nonsteroidal anti-inflammatory drugs (NSAIDs). Additionally, the IL-10 expression constructs or microparticles containing the IL-10 expression constructs used in the methods of the present invention may be co-administered with cells, such as mesenchymal stem cells or other stem cells, including stem cells bioengineered to express IL-10 expression constructs. Generally, any method known in the art can be used to monitor success of treatment in humans, including both clinical and phenotypic indicators.

[0076] Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting. In the Examples, procedures that are constructively reduced to practice are described in the present tense, and procedures that have been carried out in the laboratory are set forth in the past tense.

EXAMPLES



[0077] The following examples are put forth so as 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 regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0078] Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.

Amplification and Purification of pDNA



[0079] The plasmid construct encoding for rat interleukin-10 (pDNA-IL-10F129S) has been previously described in detail in Milligan, et al., Pain 126(1-3): 294-308 (2006). In short, The plasmid consists of a 5.9 Kilobase (Kb) circular plasmid DNA containing a transcriptional cassette consisting of a cytomegalovirus enhancer/chicken beta-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 a 149 bp inverted terminal repeat sequence. An identical plasmid lacking the IL-10 gene was used as a pDNA control. Both plasmids were amplified in SURE 2 Supercompetent E. coli cells (Agilent Technologies, USA) and isolated using an endotoxin free Giga plasmid purification kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Purified, endotoxin-free plasmids were resuspended in sterile Dulbecco's PBS (DPBS, 1, 0.1 micron pore-filtered, pH 7.2, catalog # 14190-144; Gibco, Invitrogen Corp, Grand Island, NY, USA) with 3% sucrose (DPBS-3%). The DPBS-3% vehicle was prepared using molecular biology grade D (+)-sucrose (b-D-fructofuranosyl-a-D-glucopyranoside; Sigma-Aldrich, St. Louis, MO, USA) in DPBS, 0.2 um sterile filtered (pyrogen-free syringe filter unit, catalog # 25AS020AS, Life Science Products, Inc., CO, USA) and stored in sterile, 15 ml conical tubes at 4°C until the time of use.

Microparticle preparation and characterization



[0080] Microparticles were prepared using a modified double emulsion/solvent evaporation protocol (A.M. Tinsley-Bown, et al., J. of Controlled Release 66(2-3): 229-41 (2006)). Briefly, a 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 were emulsified in the PLGA solution followed by a second emulsion in a 5% (w/v) polyvinyl alcohol, 28% calcium chloride, 3% sucrose (Sigma) and 7% (v/v) ethyl acetate solution. After 4 hours of hardening in a wash solution, the resulting microparticles were collected, lyophilized and stored at 4°C. Scanning electron microscopy (SEM) was used to examine microparticle morphology. The diameters of >1000 microparticles present in 10 different images were measured with NIH ImageJ software and binned particle diameters were used to generate a normalized frequency distribution. The zeta potential of the microparticles was measured with a Nicomp 380 ZLS Zeta Potential Analyzer, and the endotoxin levels of the resultant microparticles were tested by the LAL assay, using serial dilution as a control for inhibition. The microparticles utilized exhibited a spherical and smooth morphology under SEM and a zeta-potential of -28.04 ± 2.12 mV. The microparticles exhibited a heterogeneous size distribution with an overall median diameter of 4.67 ± 0.26 µm, which is consistent with similar methods of microparticle manufacturing and the pDNA encapsulation efficiency for the particles was 55.1%.

[0081] Total pDNA encapsulation was assessed by extracting pDNA from microparticles via sodium hydroxide dissolution, measuring the absorbance at 260 nm and comparing obtained values to DNA standards at known concentrations. Final pDNA loadings were 8.78 ± 0.65 µg pDNA/mgPLGA for PLGA-pDNA-IL-10 microparticles. Aqueous extraction of pDNA was conducted by dissolving microparticles in chloroform and allowing the pDNA to migrate into aqueous buffer. The extracted pDNA was subsequently concentrated by precipitation with ethanol and re-suspended in PBS + 3% sucrose vehicle. The structural integrity of the aqueous extracted pDNA was compared against unencapsulated pDNA (which was similarly exposed to the aqueous extraction process) 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 with UV trans-illumination at 305 nm. Biological activity of aqueous extracted pDNA was assessed by lipofectamine-mediated transfection into human embryonic kindey-293 cells according to manufacturer protocols (Invitrogen) and IL-10 protein concentrations in cell culture supernatants collected 24 hours after transfection with aqueous extracted and unencapsulated pDNA were assessed by ELISA (R&D Systems). In vitro release profiling was conducted by incubating microparticles in PBS over time in a water bath at 37°C and pDNA contents in the supernatant were quantified by a PicoGreen assay (Milligan, et al., Neuron Glia Biology 2(4) 293-308 (2006)).

[0082] Agarose gel electrophoresis of aqueous extracted pDNA from microparticles compared to unencapsulated pDNA indicated that a significant amount of the relaxed and supercoiled pDNA structural integrity was preserved after encapsulation, although a slight detection of linearized pDNA and slight alterations in the migration of multimeric pDNA species were observed after encapsulation. By comparing resultant IL-10 protein expression levels in the supernatants of human embryonic kidney-293 cells 24 hours after lipofectamine-mediated transfection with dose-matched microparticle extracted or unencapsulated pDNA, it was determined that the microparticle extracted pDNA-IL-10 exhibited a 96.8% biological activity retention for the resultant production of IL-10 (data not shown). In vitro pDNA release analysis demonstrated that 30% of the pDNA was released after 3 days and steady release was achieved for greater than 75 days. This two-phase release profile is a common characteristic of macromolecule release from emulsion based PLGA microparticles, where the enhanced phase of initial pDNA release is due to an increased pDNA content on or near the surface of the microparticles which is followed by a sustained release and diffusion of pDNA from the microparticle interior (Yeo, Archive of Endotoxin Res. 27(1): 1-12 (2004)). Endotoxin levels from microparticles with and without encapsulated pDNA were below the limits of detection for the LAL assay up to a microparticle concentration of 10 mg/ml (1 mg of microparticles/well).

D- Mannose



[0083] In embodiments where an adjuvant such as D-Mannose is employed, D-mannose (catalog # M6020) can be purchased carrier free from Sigma-Aldrich (St. Louis, MO). The D-mannose is combined with 0.1% BSA in sterile saline, and administered via intr-articular injection either concurrently with the IL-10 expression construct or from one to ten day prior to administration of the IL-10 expression construct. D-mannose is typically delivered at a dosage of 2.5 µg - 500 µg per joint in canines.

Administration for Treatment of Joint Inflammation in Canines



[0084] The IL-10 expression construct (pDNA-IL-10F129S) as "naked" pDNA was administered by acute intra-articular injection to an affected joint in a series of canines. Under IV sedation and anesthetic monitoring, the canine patient's affected joint was surgically clipped to remove all fur over the affected joint and sterilized utilizing a surgical scrub, such as chlorahexidine 2%. The patient's heart rate, blood pressure, oxygen saturation, ventilation and heart rhythm was continuously monitored. A 22- or 20-gauge hypodermic needle was inserted into the synovial space. Synovial fluid was aspirated prior to administration of the pDNA to assure proper placement of the needle. The syringe containing synovial fluid aspirate was replaced while maintaining intra-articular needle placement. Once the synovial fluid was aspirated, the therapeutic anti-inflammatory composition was administered into the joint utilizing the same intra-articular needle. Up to 1 mg plasmid DNA equivalent was injected, although as little as 700 µg was found to be effective. Where the volume per joint injection during administration did exceed 1 ml, joint fluid was aspirated to compensate. Following the successful placement of the therapeutic anti-inflammatory composition within the joint space, the patient was reversed from the sedative affects and clinically monitored. Any changes to blood pressure, heart rate, oxygenation, ventilation or heart rhythm during the procedure was corrected with the proper medical treatments. Anticipated effects of sedation include bradycardia, hypotension, hypoventilation. Oxygen therapy was continuous throughout the entire procedure to maximize oxygen saturation levels. Sedative medications tailoring to individual patient and sole medications or combinations of the following:
  1. A: Dexmedetomidine 0.5mg/m2, Reversal (atipamazole 0.05-0.2mg/lb)
  2. B: Opioids -- Butorphanol(0.05-0.1mg/lb),
  3. C: Propofol - i.v. to effect
  4. D: Benzodiazepines (diazepam): 0.1-0.2mg/lb
Subjects remained at the veterinary facility for the day (less than 12 hours) and then were allowed go home.

[0085] Clinical assessments included owner Canine Brief Pain Inventory behavioral assessments, veterinarian clinical Visual Analog Scale for pain and mobility, goniometry, pharmaceutical reduction/dependency, and video and gait monitoring. Figure 1 shows results in the form of bar graphs illustrating clinical assessment of the functional improvement and pain reduction achieved treating osteoarthritis of forelimb joints in canines after administration of the therapeutic IL-10 expression constructs of the present invention. Note that administration of the IL-10 expression constructs of the present invention resulted in significant functional improvement and pain reduction, in all of walk, trot, manipulation, range of motion and functional disability, particularly at the 11-week mark.

[0086] Figure 2 shows results in the form of bar graphs illustrating owner assessment of the functional improvement and pain reduction achieved treating osteoarthritis of forelimb joints in canines after administration of the therapeutic IL-10 expression constructs of the present invention. Note that again, there was significant improvement in all parameters of activity, quality of life, rising, walking, running and climbing stairs even at 1 week.

[0087] Figure 3 shows results in the form of bar graphs illustrating clinical assessment of the functional improvement and pain reduction achieved treating osteoarthritis of forelimb joints in canines after administration of the therapeutic IL-10 expression constructs of the present invention (pooled data). The results of clinical assessment show significant positive results, particularly in pain.

[0088] Figure 4 shows results in the form of bar graphs illustrating owner assessment of the functional improvement and pain reduction achieved treating osteoarthritis of forelimb joints in canines after administration of the therapeutic IL-10 expression constructs of the present invention (pooled data). Note in these results, similar to the results in Figure 2, there was significant improvement in all parameters of activity, quality of life, rising, walking, running and climbing stairs even at 1 week.

[0089] Figure 5 shows results illustrating improvements in range of motion achieved treating osteoarthritis of forelimb joints in canines after administration of the therapeutic IL-10 expression constructs of the present invention (pooled data). Note that the change in angle degrees showed significant improvement.

[0090] The foregoing discussion 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. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.


Claims

1. A composition comprising a bacterial plasmid IL-10 expression construct, wherein the IL-10 expression construct comprises a bacterial backbone and a nucleic acid sequence encoding interleukin-10 for use in a method of treating inflammatory joint disease in a subject, said method comprising injecting the composition into an inflamed joint of the subject.
 
2. The composition for use according to claim 1, wherein in the method the IL-10 expression construct is administered with an adjuvant.
 
3. The composition for use according to claim 2, wherein the adjuvant is selected from D-mannose, sucrose, glucose, calcium phosphate, dendrimers, oligonucleotides, high molecular weight hyaluronic acid, or liposomes.
 
4. The composition for use according to any one of the preceding claims, wherein the nucleic acid sequence encoding interleukin-10 has an amino acid substitution of serine, alanine, threonine or cysteine for wildtype phenylalanine at amino acid position 129.
 
5. The composition for use according to any one of the preceding claims, wherein the nucleic acid sequence encoding interleukin-10 encodes IL-10F129S.
 
6. The composition for use according to any one of the preceding claims, wherein the expression construct comprises at least one nuclear targeting sequence 5' to the IL-10 coding sequence.
 
7. The composition for use according to any one of the preceding claims, wherein the expression construct comprises at least one nuclear targeting sequence 3 ' to the IL-10 coding sequence.
 
8. The composition for use according to any one of the preceding claims, wherein the inflammatory joint disease is arthritis, tendonitis, bursitis, inflammation of the ligament, synovitis, gout, and systemic lupus erythematosus.
 
9. The composition for use according to any one of the preceding claims, wherein the composition comprises microparticles encapsulating the expression construct.
 
10. The composition for use according to claim 9, wherein the microparticles comprise a polymer that comprises poly(lactic-co-glycolic acid).
 
11. The composition for use according to claim 9, wherein the microparticles comprise a polymer that comprises 50:50 poly(lactic-co- glycolic acid).
 
12. The composition for use according to any one of the preceding claims, wherein the method comprises administering a composition comprising 1-1000 µg of a bacterial IL-10 expression construct and 5-1000 µg D-mannose.
 
13. The composition for use according to any one of the preceding claims, wherein in the method the D-mannose is administered concurrently with the IL-10 expression construct.
 
14. The composition for use according to any one of the preceding claims, wherein in the method the D-mannose is administered up to ten days prior to administration of the IL-10 expression construct.
 
15. The composition for use according to any one of the preceding claims, wherein the plasmid DNA comprises at least one nuclear targeting sequence both 5' and 3' to the IL-10 coding sequence.
 


Ansprüche

1. Zusammensetzung, umfassend ein Bakterienplasmid-IL-10-Expressionskonstrukt, wobei das IL-10-Expressionskonstrukt eine Bakterienhauptkette und eine Nucleinsäuresequenz umfasst, die für Interleukin-10 kodiert, zur Verwendung in einem Verfahren zur Behandlung von Gelenkentzündungskrankheiten bei einem Individuum, wobei das Verfahren das Injizieren der Zusammensetzung in ein entzündetes Gelenk des Individuums umfasst.
 
2. Zusammensetzung zur Verwendung nach Anspruch 1, wobei das IL-10-Expressionskonstrukt in dem Verfahren mit einem Adjuvans verabreicht wird.
 
3. Zusammensetzung zur Verwendung nach Anspruch 2, wobei das Adjuvans aus D-Mannose, Saccharose, Glucose, Calciumphosphat, Dendrimeren, Oligonucleotiden, hochmolekularer Hyaluronsäure oder Liposomen ausgewählt ist.
 
4. Zusammensetzung zur Verwendung nach einem der vorangegangenen Ansprüche, wobei die für Interleukin-10 kodierende Nucleinsäuresequenz eine Aminosäuresubstitution von Serin, Alanin, Threonin oder Cystein für Wildtyp-Phenylalanin an Aminosäureposition 129 aufweist.
 
5. Zusammensetzung zur Verwendung nach einem der vorangegangenen Ansprüche, wobei die für Interleukin-10 kodierende Nucleinsäuresequenz für IL-10F129S kodiert.
 
6. Zusammensetzung zur Verwendung nach einem der vorangegangenen Ansprüche, wobei das Expressionskonstrukt zumindest eine Kern-Targeting-Sequenz 5' zur für IL-10 kodierenden Sequenz umfasst.
 
7. Zusammensetzung zur Verwendung nach einem der vorangegangenen Ansprüche, wobei das Expressionskonstrukt zumindest eine Kern-Targeting-Sequenz 3' zur für IL-10 kodierenden Sequenz umfasst.
 
8. Zusammensetzung zur Verwendung nach einem der vorangegangenen Ansprüche, wobei die Gelenksentzündungskrankheit Arthritis, Tendinitis, Bursitis, Ligamententzündung, Synovitis, Gicht und systemischer Lupus erythematodes ist.
 
9. Zusammensetzung zur Verwendung nach einem der vorangegangenen Ansprüche, wobei die Zusammensetzung Mikroteilchen umfasst, die das Expressionskonstrukt einkapseln.
 
10. Zusammensetzung zur Verwendung nach Anspruch 9, wobei die Mikroteilchen ein Polymer umfassen, das Poly(milch-co-glykolsäure) umfasst.
 
11. Zusammensetzung zur Verwendung nach Anspruch 9, wobei die Mikroteilchen ein Polymer umfassen, das 50:50 Poly(milch-co-glykolsäure) umfasst.
 
12. Zusammensetzung zur Verwendung nach einem der vorangegangenen Ansprüche, wobei das Verfahren das Verabreichen einer Zusammensetzung, umfassend 1-1000 µg eines Bakterien-IL-10-Expressionskonstrukts und 5-1000 µg D-Mannose, umfasst.
 
13. Zusammensetzung zur Verwendung nach einem der vorangegangenen Ansprüche, wobei in dem Verfahren D-Mannose gleichzeitig mit dem IL-10-Expressionskonstrukt verabreicht wird.
 
14. Zusammensetzung zur Verwendung nach einem der vorangegangenen Ansprüche, wobei in dem Verfahren D-Mannose bis zu zehn Tage vor der Verabreichung des IL-10-Expressionskonstrukts verabreicht wird.
 
15. Zusammensetzung zur Verwendung nach einem der vorangegangenen Ansprüche, wobei die Plasmid-DNA zumindest eine Kern-Targeting-Sequenz sowohl 5' als auch 3' zur für IL-10 kodierenden Sequenz umfasst.
 


Revendications

1. Composition comprenant un produit de recombinaison d'expression d'IL-10 plasmidique bactérien, où le produit de recombinaison d'expression d'IL-10 comprend un squelette bactérien et une séquence d'acide nucléique codant pour l'interleukine-10, destinée à être utilisée dans un procédé de traitement d'une maladie articulaire inflammatoire chez un sujet, ledit procédé comprenant l'injection de la composition dans une articulation enflammée du sujet.
 
2. Composition destinée à être utilisée selon la revendication 1 où, dans le procédé, le produit de recombinaison d'expression d'IL-10 est administré avec un adjuvant.
 
3. Composition destinée à être utilisée selon la revendication 2, dans laquelle l'adjuvant est sélectionné parmi le D-mannose, le saccharose, le glucose, le phosphate de calcium, des dendrimères, des oligonucléotides, l'acide hyaluronique de haut poids moléculaire, ou des liposomes.
 
4. Composition destinée à être utilisée selon l'une quelconque des revendications précédentes, dans laquelle la séquence d'acide nucléique codant pour l'interleukine-10 comporte une substitution d'acide aminé sérine, alanine, thréonine ou cystéine à la phénylalanine de type sauvage à la position d'acide aminé 129.
 
5. Composition destinée à être utilisée selon l'une quelconque des revendications précédentes, dans laquelle la séquence d'acide nucléique codant pour l'interleukine-10 code pour l'IL-10F129S.
 
6. Composition destinée à être utilisée selon l'une quelconque des revendications précédentes, dans laquelle le produit de recombinaison d'expression comprend au moins une séquence de ciblage nucléaire en 5' de la séquence codant pour l'IL-10.
 
7. Composition destinée à être utilisée selon l'une quelconque des revendications précédentes, dans laquelle le produit de recombinaison d'expression comprend au moins une séquence de ciblage nucléaire en 3' de la séquence codant pour l'IL-10.
 
8. Composition destinée à être utilisée selon l'une quelconque des revendications précédentes, où la maladie articulaire inflammatoire est l'arthrite, la tendinite, la bursite, une inflammation du ligament, la synovite, la goutte, et le lupus érythémateux systémique.
 
9. Composition destinée à être utilisée selon l'une quelconque des revendications précédentes, où la composition comprend des microparticules encapsulant le produit de recombinaison d'expression.
 
10. Composition destinée à être utilisée selon la revendication 9, dans laquelle les microparticules comprennent un polymère qui comprend de l'acide poly(lactique-co-glycolique).
 
11. Composition destinée à être utilisée selon la revendication 9, dans laquelle les microparticules comprennent un polymère qui comprend de l'acide poly(lactique-co-glycolique) à 50:50.
 
12. Composition destinée à être utilisée selon l'une quelconque des revendications précédentes, où le procédé comprend l'administration d'une composition comprenant 1-1000 µg d'un produit de recombinaison d'expression d'IL-10 bactérien et 5-1000 µg de D-mannose.
 
13. Composition destinée à être utilisée selon l'une quelconque des revendications précédentes où, dans le procédé, le D-mannose est administré de manière concomitante avec le produit de recombinaison d'expression d'IL-10.
 
14. Composition destinée à être utilisée selon l'une quelconque des revendications précédentes où, dans le procédé, le D-mannose est administré jusqu'à dix jours avant l'administration du produit de recombinaison d'expression d'IL-10.
 
15. Composition destinée à être utilisée selon l'une quelconque des revendications précédentes, dans laquelle l'ADN plasmidique comprend au moins une séquence de ciblage nucléaire à la fois en 5' et en 3' de la séquence codant pour l'IL-10.
 




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REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description




Non-patent literature cited in the description