An adenovirus-based biological delivery and expression system in an effective dose for use in the treatment of osteoarthritis in humans, and a composition containing the same.

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 62 / 966,632, filed on 28 January 2020, and U.S. Provisional Patent Application No. 62 / 902,041, filed on 18 September 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Inclusion by referencing the sequence list This application includes a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. Created on September 16, 2020, the ASCII copy is filenamed "FLEX-011_001WO_Sequence Listing.txt" and is 117 kilobytes in size. [Background technology]

[0003] Osteoarthritis (OA) is a degenerative joint disease affecting the joints of humans or mammals and constitutes a significant economic and medical problem (Matthews, GL, and Hunter, DJ (2011). Emerging drugs for osteoarthritis. Expert Opin. Emerging Drugs 1-13; Brooks PM. Impact of osteoarthritis on individuals and society: how much disability? Social consequences and health economic implications. Curr Opin Rheumatol 2002; 14: 573-577). Cartilage is a tough connective tissue that covers the ends of bones in a joint. It provides a very smooth surface with relatively little friction between the hard bones, allowing for smooth movement. The onset of OA begins with inflammation, resulting from abnormal or excessive wear, followed by partial or complete loss of cartilage, leading to exposed ends that wear down against each other, resulting in pain, swelling, or loss of motor function. Currently, the detailed reasons for the initial cartilage loss that leads to osteoarthritis (OA) are unknown, but there is a strong correlation between its occurrence and joint overuse such as age, obesity, and excessive strenuous exercise.

[0004] While there are estimated to be over 100 types of arthritis, osteoarthritis (OA) is the most common form, affecting 32.5 million adults in the United States. The high prevalence of arthritis manifests itself in enormous social and personal costs.

[0005] In humans and other mammalian species, there are currently no curative treatments available for osteoarthritis (OA). Most medical treatments aim to alleviate symptoms using analgesics rather than to re-establish worn cartilage. Analgesic treatments typically involve the administration of steroids and non-steroidal anti-inflammatory drugs (NSAIDS), which have proven effective in treating OA for decades.

[0006] Further existing treatments for OA include the administration of hyaluronic acid to restore joint viscoelasticity and lubrication. Polysulfated glycosaminoglycans injected into the joint or muscle, as well as orally administered glucosamine and chondroitin sulfate, have also shown some efficacy in the treatment of OA. However, the mechanisms of action of these various treatments are not fully understood. Thus, these currently used therapies have shown limited efficacy in the treatment of OA, and the success of treatment often depends on the severity of the case. Furthermore, these drugs must be administered frequently, and sometimes in combination with each other. Such frequent drug injections into the joints are laborious, carry a risk of infection, are stressful for the patient, and are costly. Moreover, surgical treatment for OA is generally less effective and is typically performed only in patients in the advanced stages of severe disease. Therefore, there is a clear and unmet medical need for a more effective, sustained, and cost-effective treatment for OA. This disclosure addresses this need. [Overview of the project]

[0007] This disclosure includes an adenovirus-based biological delivery and expression system for treating osteoarthritis or osteoarthritis conditions in human joints, or for the prevention of such conditions in humans identified as being at risk of developing osteoarthritis or osteoarthritis conditions, comprising a genome copy (GC) of a helper-dependent adenovirus vector containing a nucleic acid sequence encoding a human interleukin-1 receptor antagonist (IL-1Ra) protein, left and right reverse terminal repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence. A pharmaceutical composition in which the expression of the human IL-1Ra gene is regulated by an NF-κB-inducible promoter located upstream of the reading frame of the nucleic acid sequence encoding the human IL-1Ra protein, and the nucleic acid sequence of an adenovirus-based biological delivery and expression system, which includes the promoter, the nucleic acid sequence encoding IL-1Ra, left and right reverse terminal repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence, may be at least 95% homologous to the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7, and the adenovirus-based biological delivery and expression system is 1.4 × 10⁶ per milliliter 8 ~1.4×10 12 The present invention provides a pharmaceutical composition containing a GC (GC / ml) helper-dependent adenovirus vector.

[0008] The nucleic acid sequence of the adenovirus-based biological delivery and expression system, including the promoter, the nucleic acid sequence encoding IL-1Ra, the left and right reverse terminal repeats, the adenovirus packaging signal, and the non-viral, non-coding stuffer nucleic acid sequence, may be at least 99% homologous to the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7.

[0009] The nucleic acid sequence of the adenovirus-based biological delivery and expression system, which includes a promoter, a nucleic acid sequence encoding IL-1Ra, left and right reverse terminal repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence, may also include the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7.

[0010] In some embodiments, the nucleic acid sequence of the adenovirus-based biological delivery and expression system, comprising a promoter, a nucleic acid sequence encoding IL-1Ra, left and right reverse-ended repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence, may be at least 95% homologous to the nucleic acid sequence of Sequence ID No. 7. In some embodiments, the nucleic acid sequence of the adenovirus-based biological delivery and expression system, comprising a promoter, a nucleic acid sequence encoding IL-1Ra, left and right reverse-ended repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence, may include the nucleic acid sequence of Sequence ID No. 7.

[0011] The nucleic acid sequence encoding IL-1Ra in the nucleic acid sequence of an adenovirus-based biological delivery and expression system may include the nucleic acid of SEQ ID NO: 4.

[0012] The nucleic acid described in SEQ ID NO: 4 expresses a human IL-1Ra protein with an amino acid sequence that is at least 95% homologous to SEQ ID NO: 6.

[0013] Adenovirus-based biological delivery and expression systems are a) 1.4 × 10 9 ~1.4×10 12 b) 1.4 × 10 9 ~1.4×10 11 ; or c) 1.4 × 10 9~1.4×10 10 It may contain 0 to 1.4×10

[0014] The adenovirus-based biological delivery and expression system may contain 1.4×10 9 ~5.6×10 9 It may contain 1.4×10 to 5.6×10 GC / ml. The adenovirus-based biological delivery and expression system may contain 1.4×10 10 ~5.6×10 10 It may contain 1.4×10 to 5.6×10 GC / ml. The adenovirus-based biological delivery and expression system may contain 1.4×10 11 ~5.6×10 11 It may contain 1.4×10 to 5.6×10 GC / ml.

[0015] The adenovirus-based biological delivery and expression system may contain 2.8×10 9 It may contain up to 2.8×10 GC / ml. The adenovirus-based biological delivery and expression system may contain 2.8×10 10 It may contain up to 2.8×10 GC / ml. The adenovirus-based biological delivery and expression system may contain 2.8×10 11 It may contain up to 2.8×10 GC / ml.

[0016] The adenovirus-based biological delivery and expression system may contain a dose volume of up to 5 ml.

[0017] The adenovirus-based biological delivery and expression system may contain a total dose of 7×10 9 ~2.8×10 10 It may contain a total dose of 7×10 to 2.8×10 GC. The adenovirus-based biological delivery and expression system may contain 7×10 10 ~2.8×10 11 It may contain a total dose of 7×10 to 2.8×10 GC. The adenovirus-based biological delivery and expression system may contain 7×10 11 ~2.8×10 12 It may contain a total dose of 7×10 to 2.8×10 GC.

[0018] The adenovirus-based biological delivery and expression system may contain a total dose of 1.4×10 10 It may contain a total dose of 1.4×10 GC. The adenovirus-based biological delivery and expression system may contain 1.4×10 11The total dose of GC may be included. The adenovirus-based biological delivery and expression system is 1.4 × 10⁻⁶. 12 The total dose of GC may be included.

[0019] The pharmaceutical composition can be formulated for intra-articular, intratendinous, intramuscular, or subacromial injection into a human joint. In a preferred embodiment, the pharmaceutical composition can be formulated for intra-articular injection into a human joint.

[0020] This disclosure provides a method for infecting articular cells of one or more osteoarthritis-affected joints of a human suffering from osteoarthritis or an osteoarthritis-affected condition with an adenovirus-based biological delivery and expression system, comprising: a) infecting articular cells of an osteoarthritis-affected joint of a human requiring such treatment with a pharmaceutical composition comprising the adenovirus-based biological delivery and expression system disclosed herein; and b) expressing IL-1Ra in a target region within the osteoarthritis-affected joint.

[0021] Articular cells can be infected once with an adenovirus-based biological delivery and expression system. Articular cells can be infected two or more times with an adenovirus-based biological delivery and expression system.

[0022] In one embodiment, when articular cells are infected with an adenovirus-based biological delivery and expression system two or more times, each infection includes a helper-dependent adenovirus vector with the same number of genomic copies. In another embodiment, when articular cells are infected with an adenovirus-based biological delivery and expression system two or more times, each infection includes a helper-dependent adenovirus vector with a different number of genomic copies.

[0023] When articular cells are infected with an adenovirus-based biological delivery and expression system more than once, each infection takes place in the same osteoarthritis-affected joint of a human. When articular cells are infected with an adenovirus-based biological delivery and expression system more than once, all second and subsequent infections take place in a different osteoarthritis-affected joint of a human than the one in which the previous infection occurred.

[0024] In some embodiments, arthrocyte infection may include intra-articular, intratendinous, intramuscular, or subacromial injection. In preferred embodiments, arthrocyte infection may include intra-articular injection.

[0025] The method may further include the step of (c) treating or monitoring the progression of osteoarthritis or an osteoarthritis-related condition in an osteoarthritis-affected joint by expressing IL-1Ra in a target region within the osteoarthritis-affected joint.

[0026] The method may further include (d) a step of continuing to administer an adenovirus-based biological delivery and expression system to a human joint affected by osteoarthritis if a step of monitoring the treatment or progression of osteoarthritis or an osteoarthritis condition in an affected joint indicates that osteoarthritis or an osteoarthritis condition in the human joint is not under control or treatment; or (e) a step of further adjusting the genomic copy number of the helper-dependent adenovirus vector in an adenovirus-based biological delivery and expression system and administering it to a human joint affected by osteoarthritis if a step of monitoring the treatment or progression of osteoarthritis or an osteoarthritis condition in an affected joint indicates that osteoarthritis or an osteoarthritis condition in the human joint has progressed.

[0027] This disclosure relates to (a) culturing and continuously expanding host cells; (b) infecting the continuously expanded host cells from (a) with the helper-dependent adenovirus (HDAd) and helper virus of the present invention; (c) culturing the infected cells from (b); (d) harvesting and lysing the infected cells from (c) to produce cell lysates; (e) digesting the host cell DNA in the cell lysates from (d); (f) clarifying the cell lysates from (e); (g) ultracentrifugation of the clarified cell lysates from (f); (h) collecting the virus from the ultracentrifuged cell lysates from (g); (i) gradient ultracentrifugation of the virus sample from (h); and (j) gradient ultracentrifugation of (i). A process is provided for producing the pharmaceutical composition according to claim 1, comprising: collecting a virus from an ultracentrifugally separated virus sample; performing ultracentrifugation of the virus sample of (k)(j) at the same density; collecting a virus from the ultracentrifugally separated virus sample of (l)(k) at the same density; performing ultracentrifugation of the virus sample of (m)(l) at the same density; collecting a virus from the ultracentrifugally separated virus sample of (n)(m) at the same density; dialysis of the collected virus of (o)(n); collecting and diluting the dialyzed virus of (p)(o); formulating the diluted virus of (q)(p); and filtering the formulated virus of (r)(o).

[0028] This disclosure relates to an adenovirus-based biological delivery and expression system for the treatment of osteoarthritis or osteoarthritis conditions in human joints, or for the prevention of such conditions in humans identified as being at risk of developing osteoarthritis or osteoarthritis conditions, wherein the adenovirus-based biological delivery and expression system comprises a helper-dependent adenovirus vector genome copy (GC) containing a nucleic acid sequence encoding a human interleukin-1 receptor antagonist (IL-1Ra), left and right reverse terminal repeats, an adenovirus packaging signal and a non-viral, non-coding stuffer nucleic acid sequence, and the expression of the human IL-1Ra gene is carried out by the nucleic acid sequence encoding the human IL-1Ra protein The nucleic acid sequence of the adenovirus-based biological delivery and expression system, located upstream of the column's reading frame and regulated by an NF-κB-inducible promoter, including the promoter, the nucleic acid sequence encoding IL-1Ra, left and right reverse terminal repeats, adenovirus packaging signal and non-viral, non-coding stuffer nucleic acid sequences, may be at least 95% homologous to the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7, and the adenovirus-based biological delivery and expression system is isolated from a helper-dependent adenovirus vector and host cells infected with the helper virus, and the adenovirus-based biological delivery and expression system a) 1.4 × 10⁶ per milliliter of synovial fluid in human joints 8 ~1.4×10 12 a) Helper-dependent adenovirus vector of GC (GC / ml); b) less than 15% helper virus particles; c) less than 10% empty capsid; d) host cell protein less than 100 μg / ml; e) host cell nucleic acid less than 20 ng / ml; f) endotoxin less than 35 EU / ml; and g) 300 GC / TCID 50 This provides an adenovirus-based biological delivery and expression system, including the following ratios of viral particles to infectious units.

[0029] Any of the above embodiments can be combined with any other embodiment.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the field to which this disclosure belongs. In this specification, unless the context explicitly indicates otherwise, the singular form includes the plural; for example, the terms “a,” “an,” and “the” are understood to be singular or plural, and the term “or” is understood to be inclusive. For example, “an element” means one or more elements. Throughout this specification, variations of the word such as “comprising” or “comprises” or “comprising” will be understood to mean the inclusion of an element, integer, or process, or a group of elements, integers, or processes, being described, but not the exclusion of any other element, integer, or process, or a group of elements, integers, or processes. "Approximately" can be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clearly indicated by the context, all numerical values ​​provided herein are modified by the term "approximately."

[0031] Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. References cited herein are not acknowledged as prior art to the claimed disclosure. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not intended to be limiting. Other features and advantages of this disclosure are evident from the detailed description and claims below. [Brief explanation of the drawing]

[0032] [Figure 1]Figure 1 depicts the genome map of the humantakinogene Hadenovec (FX201). ITR = reverse terminal repeat (1-103 bp on the 5' side; 29,158-29,260 bp on the 3' side), Ψ = packaging signal (240-375 bp), HPRT stuffer = human hypoxanthine phosphoribosyltransferase (463-16,518 bp), human cosmid insert = human cosmid (16,532-27,637 bp), SV40 polyA = monkey virus 40 polyA (27,750-28,020 bp), huIL-1Ra = human interleukin-1 receptor antagonist, target genome (28,033-28,566 bp), NF-κB5-ELAM promoter = ινδυχιβλε promoter (28,581-28,842 bp). [Figure 2] Figure 2 depicts the basic gene map of the helper-dependent adenovirus vector of this disclosure. The vector skeleton consists of left and right reverse terminal repeats (ITRs), an adenovirus packaging signal (Ψ), and non-coding, non-viral stuffer sequences (the remaining unmarked sequences between the ITRs). The cDNA of equine IL-1Ra (GQ-201), mouse IL-1Ra, or human IL-1Ra is cloned between the left and right ITRs of the virus in the adenovirus vector used. The IL-1Ra gene is controlled by an inflammation-sensitive NF-KB5-ELAM promoter. [Figure 3A] Figures 3A and 3B depict the transduction efficiency of HDAd and AAV vectors in mouse joints. Figure 3A depicts a comparison of representative joints injected with HDAd-GFP and AAV6-GFP, which were considered to be the AAV serotypes exhibiting the most potent GFP expression (top image: fluorescence; bottom image: corresponding bright-field image). Figure 3B depicts a comparison of HDAd-GFP with all AAV serotypes. Images of individual joints from two mice per group are shown. [Figure 3B]Figures 3A and 3B depict the transduction efficiency of HDAd and AAV vectors in mouse joints. Figure 3A depicts a comparison of representative joints injected with HDAd-GFP and AAV6-GFP, which were considered to be the AAV serotypes exhibiting the most potent GFP expression (top image: fluorescence; bottom image: corresponding bright-field image). Figure 3B depicts a comparison of HDAd-GFP with all AAV serotypes. Images of individual joints from two mice per group are shown. [Figure 4A] Figures 4A and 4B depict a comparison of marker gene expression levels and durations between helper-dependent adenovirus vectors and first-generation adenovirus vectors. Figure 4A depicts bioluminescence imaging of mice infected with helper-dependent adenovirus vectors and first-generation adenovirus vectors, showing that both vectors mediate similar levels of marker gene expression. Representative images of two mice from each group are shown. Figure 4B depicts luciferase expression in the mice described in Figure 4A, followed by repeated bioluminescence imaging and quantification. [Figure 4B] Figures 4A and 4B depict a comparison of marker gene expression levels and durations between helper-dependent adenovirus vectors and first-generation adenovirus vectors. Figure 4A depicts bioluminescence imaging of mice infected with helper-dependent adenovirus vectors and first-generation adenovirus vectors, showing that both vectors mediate similar levels of marker gene expression. Representative images of two mice from each group are shown. Figure 4B depicts luciferase expression in the mice described in Figure 4A, followed by repeated bioluminescence imaging and quantification. [Figure 5]Figures 5A and 5B illustrate how a helper-dependent adenovirus vector efficiently infects synovial and chondrocyte cells. Mice were intra-articularly injected with HDAd expressing 108-109VP LacZ. One day later, mice were sacrificed, and LacZ staining was performed on sectioned joints. Figures 5A and 5B depict photographs of LacZ expression on sectioned joints of mice infected with HDAd expressing 108VP and 109VP LacZ, respectively, showing low-magnification photographs (top panel) and high-magnification (bottom panel) photographs (40x) of the framed area in Figure 5B (left photograph) (5x). S represents synovial membrane, C represents chondrocytes, and the thick, dark lines indicated with arrowheads depict LacZ staining. [Figure 6] Figure 6 illustrates the production of large amounts of IL-1Ra by cells infected with HDAd-IL-1Ra. The y-axis depicts the levels of IL-1Ra measured by ELISA performed using cell culture supernatants of human embryonic kidney cells (HEK293) infected with or without LPS stimulation, HDAd-IL-1Ra, HDAd-GFP, or a mock, as shown. The x-axis depicts the time period for IL-1Ra measurement. Each data point represents three independent experiments, and the error bars indicate standard deviation (SD). [Figure 7] Figure 7 illustrates that HDAd-IL-1Ra prevents the development of osteoarthritis (OA) in mice. The Y-axis depicts the level of OA as assessed by a blinded pathologist according to OARSI (International Osteoarthritis Society) criteria (assigning scores on a scale of 1-6, 1: no signs of OA, 6: maximum OA). The X-axis depicts the three treatment groups: HDAd-IL-1Ra, HDAd-GFP, or mock. * indicates a statistically significant difference: p<0.05 by one-way ANOVA; n=10 joints per group. [Figure 8]Figures 8A–8C depict the histopathological evaluation of osteoarthritis (OA) in mice. Figure 8A depicts the OARSI score, Figure 8B depicts the synovitis score, and Figure 8C depicts the percentage of joints with osteophytes in mice treated with HDAd-mIL-1Ra, HDAd-GFP, or the medium. Mean ± SD and individual knee data are shown. *P<0.05; One-way ANOVA with Tukey's multiple comparison test. [Figure 9A] Figures 9A–9C illustrate how HDAd-IL-1Ra effectively treats osteoarthritis (OA) in mice. Figure 9A depicts the OA scores of joints treated with HDAd-IL-1Ra compared to controls. OA scores for mice intra-articularly injected with 108VP of HDAd-IL-1Ra, HDAd-GFP, or a mock are plotted on the y-axis. Blinded pathologists assessed the level of OA according to OARSI (International Osteoarthritis Society) criteria (assigning scores on a scale of 1–6, 1: no signs of OA, 6: maximum OA). * indicates a statistically significant difference: p<0.05 by one-way ANOVA; n=8 joints per group. Figure 9B depicts the cartilage volume of joints treated with HDAd-IL-1Ra compared to controls. * indicates a statistically significant difference: p<0.05 by one-way ANOVA; n=6 joints per group. Figure 9C depicts the cartilage surface area of ​​joints treated with HDAd-IL-1Ra compared to the control. * indicates a statistically significant difference: p<0.05 by one-way ANOVA; n=6 joints per group. [Figure 9B]Figures 9A–9C illustrate how HDAd-IL-1Ra effectively treats osteoarthritis (OA) in mice. Figure 9A depicts the OA scores of joints treated with HDAd-IL-1Ra compared to controls. OA scores for mice intra-articularly injected with 108VP of HDAd-IL-1Ra, HDAd-GFP, or a mock are plotted on the y-axis. Blinded pathologists assessed the level of OA according to OARSI (International Osteoarthritis Society) criteria (assigning scores on a scale of 1–6, 1: no signs of OA, 6: maximum OA). * indicates a statistically significant difference: p<0.05 by one-way ANOVA; n=8 joints per group. Figure 9B depicts the cartilage volume of joints treated with HDAd-IL-1Ra compared to controls. * indicates a statistically significant difference: p<0.05 by one-way ANOVA; n=6 joints per group. Figure 9C depicts the cartilage surface area of ​​joints treated with HDAd-IL-1Ra compared to the control. * indicates a statistically significant difference: p<0.05 by one-way ANOVA; n=6 joints per group. [Figure 9C] Figures 9A–9C illustrate how HDAd-IL-1Ra effectively treats osteoarthritis (OA) in mice. Figure 9A depicts the OA scores of joints treated with HDAd-IL-1Ra compared to controls. OA scores for mice intra-articularly injected with 108VP of HDAd-IL-1Ra, HDAd-GFP, or a mock are plotted on the y-axis. Blinded pathologists assessed the level of OA according to OARSI (International Osteoarthritis Society) criteria (assigning scores on a scale of 1–6, 1: no signs of OA, 6: maximum OA). * indicates a statistically significant difference: p<0.05 by one-way ANOVA; n=8 joints per group. Figure 9B depicts the cartilage volume of joints treated with HDAd-IL-1Ra compared to controls. * indicates a statistically significant difference: p<0.05 by one-way ANOVA; n=6 joints per group. Figure 9C depicts the cartilage surface area of ​​joints treated with HDAd-IL-1Ra compared to the control. * indicates a statistically significant difference: p<0.05 by one-way ANOVA; n=6 joints per group. [Figure 10]Figures 10A and 10B depict the evaluation of therapeutic efficacy using microcomputed tomography. Figure 10A depicts changes in cartilage volume, and Figure 10B depicts changes in cartilage-covered bone regions as assessed by micro-CT imaging, in mice with pre-established OA who received IA administration of HDAd-mIL-1Ra, HDAd-GFP, or the medium two weeks after surgery, as well as in age-matched mice that had not undergone CLT surgery. Mean ± SD and individual joint values ​​are shown. *P<0.05; One-way ANOVA with Tukey's multiple comparison test. [Figure 11] Figure 11 depicts the composite (total) scores by group for cartilage / bone assessment in sham and ACLT-surgered rats regarding the OARSI score for cartilage / bone. The y-axis shows the total OARSI composite score, and the x-axis shows the different treatments and doses. The dashed line shows the mean; the solid line shows the median; the dots show outliers; the tips of the boxes show the first quartile (bottom) and third quartile (top); the ends of the whiskers show the minimum and maximum values ​​for ACLT, anterior cruciate ligament dissection; Min, minimum; Max, maximum; VP, viral particle. [Figure 12A] Figures 12A–12D depict the group total scores for (A) structural changes, (B) SOFG staining loss, (C) clonal formation, and (D) chondrocyte loss in sham and ACLT-surgered rats. The y-axis shows the total OARSI subscore, and the x-axis shows the different treatments and doses. The dashed line shows the mean; the solid line shows the median; the dots show outliers; the tips of the boxes show the first quartile (bottom) and third quartile (top); the ends of the whiskers show the minimum and maximum values ​​for ACLT, anterior cruciate ligament transection; Min, minimum; Max, maximum; VP, viral particle. [Figure 12B]Figures 12A–12D depict the group total scores for (A) structural changes, (B) SOFG staining loss, (C) clonal formation, and (D) chondrocyte loss in sham and ACLT-surgered rats. The y-axis shows the total OARSI subscore, and the x-axis shows the different treatments and doses. The dashed line shows the mean; the solid line shows the median; the dots show outliers; the tips of the boxes show the first quartile (bottom) and third quartile (top); the ends of the whiskers show the minimum and maximum values ​​for ACLT, anterior cruciate ligament transection; Min, minimum; Max, maximum; VP, viral particle. [Figure 12C] Figures 12A–12D depict the group total scores for (A) structural changes, (B) SOFG staining loss, (C) clonal formation, and (D) chondrocyte loss in sham and ACLT-surgered rats. The y-axis shows the total OARSI subscore, and the x-axis shows the different treatments and doses. The dashed line shows the mean; the solid line shows the median; the dots show outliers; the tips of the boxes show the first quartile (bottom) and third quartile (top); the ends of the whiskers show the minimum and maximum values ​​for ACLT, anterior cruciate ligament transection; Min, minimum; Max, maximum; VP, viral particle. [Figure 12D] Figures 12A–12D depict the group total scores for (A) structural changes, (B) SOFG staining loss, (C) clonal formation, and (D) chondrocyte loss in sham and ACLT-surgered rats. The y-axis shows the total OARSI subscore, and the x-axis shows the different treatments and doses. The dashed line shows the mean; the solid line shows the median; the dots show outliers; the tips of the boxes show the first quartile (bottom) and third quartile (top); the ends of the whiskers show the minimum and maximum values ​​for ACLT, anterior cruciate ligament transection; Min, minimum; Max, maximum; VP, viral particle. [Figure 13]Figure 13 depicts the composite (total) scores by group for synovial evaluation in sham and ACLT-surgered rats by H&E staining. The y-axis shows the total score, and the x-axis shows the different treatments and doses. The dashed line shows the mean; the solid line shows the median; the dots show outliers; the tips of the boxes show the first quartile (bottom) and third quartile (top); the ends of the whiskers show the minimum and maximum values ​​for ACLT, anterior cruciate ligament transection; Min, minimum; Max, maximum; VP, viral particle. [Figure 14] Figure 14 depicts the in vitro expression of equine IL-1Ra after infection with HDAd-eqIL-1Ra. The concentrations of equine IL-1Ra in the supernatant of cell cultures infected with different concentrations of HDAd-eqIL-1Ra (GQ-201) or HDAd-GFP are shown. Group "B" was incubated with 100 μg / mL LPS from day 3 to day 4. Mean ± SEM values ​​are shown. [Figure 15] Figure 15 depicts synovial fluid levels of IL-1Ra. Equine synovial fluid IL-1Ra levels were measured in the synovial fluid for a dose-escalation study of equine IL-1Ra delivered by helper-dependent adenovirus in equine joints. IL-1Ra in naive joints was measured only on day 90 (pre-injection) and day 92. CarpJ, carpal joint; MCPJ, metacarpophalangeal joint; MTPJ, metatarsophalangeal joint; LPS, lipopolysaccharide; NSAID, nonsteroidal anti-inflammatory drug. [Figure 16] Figure 16 illustrates the clinical scoring of OA in the equine osteochondral chip model. Figure 16 depicts the mean ± SEM of the clinical score of the injected joint at the final evaluation time of day 72, on the y axis, corresponding to the parameter shown on the x axis. The parameter "flexion" represents limping after joint flexion over 20 seconds. *P<0.05; Kruskal-Wallis test with pairwise comparison with the media group. [Figure 17]Figure 17 depicts cartilage fibrillation in treated joints in a horse osteochondral chip model. The left panel depicts the mean ± SEM of cartilage fibrillation scores in the intermediate carpal (CI), radial carpal (CR), third articular surface of the intermediate carpal (C3IF), and third articular surface of the radial carpal (C3RF) regions of joints injected with HDAd-eqIL-1Ra (GQ-201), PBS (placebo), or sham-operated joints. The right panel depicts the mean ± SEM of the total cartilage fibrillation score; the sum of individual scores. *P<0.05; Kruskal-Wallis test with pairwise comparison with the medium group. [Figure 18] Figure 18 depicts the histological analysis of treated joints in a horse osteochondral chip model. Left panel: Mean ± SEM scores for assessed parameters in joints injected with HDAd-eqIL-1Ra (GQ-201), PBS (placebo), or sham-operated joints. Right panel: Mean ± SEM total histological score; sum of individual histological scores. *P<0.05; Kruskal-Wallis test with pairwise comparison with media group. [Figure 19] Figures 19A and 19B depict graphs showing live cell density and terminal cell survival rates in adherent cultures. Figure 19A depicts live cell density, shown as cells / cm2 on the y-axis, corresponding to seeding and 5 passages (P1-P5) on the x-axis and at the end of infection (harvest), for 3 batches of FX201 and 1 batch of rat orthologue, as shown. Figure 19B depicts terminal survival rates (survival percentage) on the y-axis, measured for each of the 5 passages (P1-P5) and at the end of infection, as shown on the x-axis. Both graphs show data for all 5 passages and infections for Tox (human), Tox (rat), ENG, and GMP lots. The cell expansion was split into two streams after passage 5 for one further co-infection. The first stream of cell expansion data is shown. Cell counting was not taken before infection. The viable cell density (VCD) was calculated at the time of subculturing and quantified at the end of each subculturing. [Figure 20]Figure 20 depicts the product yield in the downstream processing steps. Virus particle (VP) production (productivity of the pre-purification batch) and yield values ​​(yield of the post-purification batch and post-purification yield %), normalized over batch size, are plotted on the y-axis for Tox (human), Tox (rat), ENG, and GMP lots as shown. Data for each lot are presented as the percentage difference from the mean of the four lots. [Figure 21] Figure 21 depicts infectivity and gene expression for different lots. Infectivity and the ratio of genome copies to infection, as measured as TCID50, as well as IL-1Ra expression, are depicted for Tox (human), Tox (rat), ENG, and GMP lots, as shown. [Modes for carrying out the invention]

[0033] This disclosure provides compositions for improved delivery and expression systems that enable long-term expression of a bioactive recombinant interleukin-1 receptor antagonist (IL-1Ra) in human joint cells for the treatment and prevention of osteoarthritis. A novel IL-1Ra gene therapy (FX201, humantakinogene hadenovec) for intra-articular (IA) administration developed for the treatment of osteoarthritis or osteoarthritis conditions in patients is disclosed herein. FX201 (humantakinogene hadenovec) is a helper-dependent adenovirus (HDAd) that delivers a nucleic acid sequence encoding human IL-1Ra under the control of a nuclear factor-κB (NF-κB) inducible promoter for IA administration to patients with osteoarthritis or osteoarthritis conditions. After IA injection, FX201 infects cells in the joint and locally produces IL-1Ra in response to inflammation. FX201 is a non-replicating, non-integrated HDAd vector that does not contain a viral coding sequence and is engineered to carry the gene coding sequence for IL-1Ra. Adenovirus packaging signals and reverse terminal repeats (ITRs) are necessary for manufacturing and therefore only they remain in the FX201 genome. Transcription is regulated by an inflammation-sensitive NF-κB-inducible promoter that drives IL-1Ra expression in response to an inflammatory environment.

[0034] FX201 can be administered as a single dose by IA injection. The expected clinical benefits are sustained symptom relief, including both pain reduction and improved or restored function, and beneficial modification of the underlying disease process in patients with osteoarthritis or osteoarthritis in the human joints. Advantageously, the adenovirus delivery and expression system of this disclosure is specifically located within the joint when administered intra-articularly. Most importantly, the vector sequence cannot be detected at measurable concentrations in the liver of mice treated with the adenovirus system of this disclosure. Therefore, while IL-1Ra concentrations are expected to be highest in the joint injected with the vector of this disclosure, no significant side effects are expected in other organs. The properties of FX201 are described below.

[0035] Vector Skeleton: FX201 is a non-replicating, non-integrated HDAd vector. Its genomic components consist of double-stranded linear DNA approximately 29.3 kilobases (kb) in size. Annotated sequences obtained by next-generation sequencing confirm key elements in the FX201 genome. The FX201 genome contains the minimum adenoviral elements necessary for amplification and packaging that enable its production: left and right reverse terminal repeats (hereinafter referred to herein as "L ITR" and "R ITR," respectively) and a packaging signal (Ψ). The approximately 1.1 kb FX201 genome consists of a nucleic acid sequence encoding human IL-1Ra, inserted in reverse (right to left) at the right end of the genome, and a promoter placed immediately before the R ITR. The promoter is a five-conserved NF-κB binding motif repeat fused to the proximal promoter region of the human ELAM gene, which responds to inflammatory cytokines (Schindler 1994). The approximately 27kb FX201 genome consists of a non-coding stuffer sequence comprising human hypoxanthine phosphoribosyltransferase (HPRT) and human cosmid inserts inserted to expand the FX201 genome to a size that allows for efficient packaging of the vector genome into each viral particle. The genome map of FX201 is shown in Figure 1.

[0036] Target gene: The FX201 genome contains a 534 base pair (bp) sequence of human IL-1Ra regulated by a 262 bp sequence of an NF-κB inducible promoter.

[0037] Gene maps of FX201, the HDAd vector of the present invention, are disclosed herein (Figure 2). All three vectors contain an inflammation-sensitive NF-κB5-ELAM promoter upstream of the IL-1Ra cDNA described in either SEQ ID NO: 1 or 4, as well as ITR and adenovirus packaging signals. The complete vector sequences of GQ-201, HDAd-mIL-Ra, and HDAd-human IL-1Ra are shown in SEQ ID NOs: 2, 3, and 7, respectively. The only difference between the three vectors is that GQ-201 carries a horse variant of IL-1Ra, HDAd-mIL-Ra carries a mouse variant of IL-1Ra, and HDAd-huIL-1Ra carries human IL-1Ra. For example, HDAd-mIL-Ra of the nucleic acid sequence described in SEQ ID NO: 3 may contain the nucleic acid encoding mouse IL-1Ra described in SEQ ID NO: 1. For example, HDAd-mIL-Ra of the nucleic acid sequence described in SEQ ID NO: 7 may contain the nucleic acid encoding mouse IL-1Ra described in SEQ ID NO: 4.

[0038] The vectors were cloned by a standard digestion / ligation reaction following the following strategy: The luciferase cDNA in pNifty-luc, a plasmid containing luciferase cDNA driven by the NF-KB5-ELAM promoter, was excised using NcoI and NheI, and the cDNA of equine or mouse IL-1Ra was ligated to this position. The NF-KB5-ELAM promoter-mouse IL-1Ra or NF-KB5-ELAM promoter-equine IL-1Ra cassette was excised using NotI and Pad or EcoRI and Pad, blunted, and linearized using Sail, and then inserted into the pLPBL shuttle plasmid. Next, the NF-KB5-ELAM promoter-mouse IL-1Ra or NF-KB5-ELAM promoter-equine IL-1Ra cassettes were excised using AscI adjacent to both sides of the multicloning site and ligated into a ρΔ28 plasmid linearized with AscI (Toietta, G., Pastore, L, Cerullo, V., Finegold, M., Beaudet, AL, and Lee, B. (2002). Generation of helper-dependent adenoviral vectors by homologous recombination. Mol Ther 5, 204-210), yielding the genomic plasmids pA28-mll-1Ra and pA28-eqll-1Ra. These plasmids were digested with Pmel to linearize the vectors, release the reverse terminal repeats, and excavate the bacterial resistance gene.The vectors were rescued and amplified using the helper virus AdNG163R-2 and 116 cell factories as previously described (Palmer, D., and Ng, P. (2003). Improved system for helper-dependent adenoviral vector production. Mol Ther 8, 846-852; Suzuki, M., Cela, R., Clarke, C, Bertin, TK, Mourino, S., and Lee, B. (2010). Large-scale production of high-quality helper-dependent adenoviral vectors using adherent cells in cell factories. Hum Gene Ther 21, 120-126).

[0039] Composition of the present disclosure The compositions of this disclosure may also include an adenovirus-based biological delivery and expression system based on a helper-dependent adenovirus vector comprising a nucleic acid sequence encoding a human or mammalian interleukin-1 receptor antagonist (IL-1Ra), L ITR, R ITR, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence.

[0040] The helper-dependent adenovirus vectors of this disclosure minimize the immune response in the host and confer long-term gene expression of human or mammalian IL-1Ra in joints affected by osteoarthritis.

[0041] In some embodiments, the sequence encoding a human or mammalian interleukin-1 receptor antagonist (IL-1Ra) in the compositions of the Disclosure can be controlled by an inflammation-sensitive promoter. Although we do not wish to be constrained by theory, since only disease-affected cells express and secrete the IL-1Ra gene product, while unaffected cells do not, the use of an inflammation-sensitive promoter in the compositions of the Disclosure provides specific control of IL-1Ra gene expression in tissue cells in an osteoarthritis state. In some embodiments, the promoter sequence may be located upstream of the reading frame of the sequence encoding human or mammalian IL-1Ra.

[0042] While we do not wish to be constrained by theory, inflammation-sensitive promoters used in the compositions of this disclosure can be specifically activated by increasing the levels of factors including inflammatory stimulants and / or cytokines. During osteoarthritis, various immunostimulants and cytokines are released, resulting in high levels of promoter activators. In non-limiting examples, the immunostimulant may be lipopolysaccharide (LPS), a major component of the outer cell membrane of Gram-negative bacteria. The released immunostimulants and / or cytokines can activate transcription factors such as NF-κB, which modulate the NF-κB promoter. Thus, the release of such osteoarthritis-specific immunostimulants and / or cytokines may enable the regulation of gene expression in human or mammalian joints for treating or preventing osteoarthritis.

[0043] Any inflammation-sensitive promoter that results in specific expression of the IL-1Ra gene product in tissues in an osteoarthritis state can be used in the context of this disclosure. Preferred inflammation-sensitive promoters for use in this disclosure include, but are not limited to, promoters that can be induced by NF-κB, interleukin-6 (IL-6), interleukin-1 (IL-1), tumor necrosis factor (TNF), cyclooxygenase-2 (COX-2), complement factor-3 (C3), serum amyloid A3 (SAA3), macrophage inflammatory protein-1a (MIP-1a), or hybrid constructs of the above.

[0044] In a preferred embodiment, the inflammation-sensitive promoter is the NF-κB5-ELAM promoter. For several reasons, an NF-κB-inducible promoter consisting of five conserved NF-κB-binding motif repeats fused to the proximal promoter region of the human endothelial leukocyte adhesion molecule (ELAM) gene was selected to drive IL-1Ra expression. Firstly, NF-κB as a transcription factor is ubiquitously expressed in all cells in the body and in any transduced cells, and in principle can express the IL-1Ra transgene when stimulated with inflammatory signals. Therefore, there are no cell-specific requirements for FX201 to induce IL-1Ra expression. Furthermore, NF-κB is a terminal signaling molecule for receptors of inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-α, as well as other immune cell receptors such as Toll-like receptors, where it acts to initiate cellular responses to many inflammatory inputs. Therefore, activation of IL-1Ra production is designed to be stimulated in the joints by various inflammatory signals.

[0045] While we do not wish to be constrained by theory, after intra-articular injection, the IL-1Ra gene is delivered to articular cells, including, but not limited to, synovial cells. Synovial cells affected by inflammation begin to produce recombinant IL-1Ra protein under the control of inflammation-sensitive promoters (e.g., NF-κB promoter). Large amounts of IL-1Ra are then secreted into the joint cavity, where IL-1Ra inhibits inflammation and halts chondrolysis by blocking interleukin-1 receptors on the surface of synovial cells and cells embedded in cartilage. Most importantly, high local concentrations of recombinant IL-1Ra do not cause any side effects.

[0046] As described herein, pain, inflammation, and chondrolysis are efficiently inhibited using the adenovirus-based biological delivery and expression system of this disclosure. High local and low systemic concentrations of the therapeutic protein IL-1Ra are achieved by administration of the compositions of this disclosure, resulting in maximum efficacy in the treatment of OA with no or minimal side effects. It is further exemplified that cells containing the helper-dependent adenovirus vector of this disclosure can produce recombinant IL-1Ra for a long period of at least 3 months, at least 6 months, or at least 1 year, whichever is more than one of these periods. As a result, the medical and economic burden associated with frequent joint injections required in known short-term treatments will be significantly reduced. Thus, potential complications associated with OA treatment will be minimized, joint health will be preserved, and sustained health improvements will be obtained in the treated animals or humans.

[0047] Furthermore, since arthrocytes infected with the adenovirus vector of this disclosure remain silent in the absence of immunostimulants that can activate the NF-κB5-ELAM promoter or any other inflammation-sensitive promoter, the helper-dependent inflammation-sensitive IL-1Ra production of the helper-dependent adenovirus vector of this disclosure enables the prevention of the development of osteoarthritis. IL-1Ra is produced and secreted only after the promoter is activated as a result of inflammation, and only when osteoarthritis has begun. Thus, by using the adenovirus delivery and expression system of this disclosure, this mechanism enables the prevention of osteoarthritis development in its early stages.

[0048] Since IL-1Ra is no longer produced once the osteoarthritis condition resolves or disappears, the inflammatory IL-1Ra production of the helper-dependent adenovirus vector of this disclosure is also safer for administration to subjects.

[0049] The helper-dependent adenoviral vectors of this disclosure do not carry any viral sequences except for the L ITR, R ITR, and adenoviral packaging signals. The preferred helper-dependent adenoviral vectors used in this disclosure are based on helper viruses and helper-dependent skeletal systems developed by Palmer and Ng (Palmer, D., and Ng, P. (2003). Improved system for helper-dependent adenoviral vector production. Mol Ther 8, 846-852) and Toietta et al (Toietta, G., Pastore, L., Cerullo, V., Finegold, M., Beaudet, AL, and Lee, B. (2002). Generation of helper-dependent adenoviral vectors by homologous recombination. Mol Ther 5, 204-210). Preferred adenovirus delivery and expression systems according to this disclosure may include nucleic acid sequences of adenovirus-based biological delivery and expression systems, or bioeffective portions thereof, as described in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7, which include a promoter, a nucleic acid sequence encoding IL-1Ra, left and right reverse terminal repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence. The nucleic acid sequence in SEQ ID NO: 2 describes a mouse helper-dependent adenovirus vector, the sequence in SEQ ID NO: 3 describes a horse helper-dependent adenovirus vector, and the sequence in SEQ ID NO: 7 describes a human helper-dependent adenovirus vector, with all three vectors each carrying either a mouse, horse IL-1Ra gene, or human IL-1Ra gene. Preferably, the systems of this disclosure have at least 96%, 97%, 98%, or 99% sequence homology with the vectors described in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7.

[0050] In the context of this disclosure, “long-term expression” means that the gene product of the adenovirus delivery and expression system (i.e., IL-1Ra) is expressed in a joint infected with the helper-dependent adenovirus vector of this disclosure for at least 3 months, 6 months, or 12 months. In a preferred embodiment, IL-1Ra is expressed in a joint infected with the helper-dependent adenovirus vector of this disclosure for at least 3 months.

[0051] In the context of this disclosure, “biologically effective” means that the gene product of the adenovirus delivery and expression system includes a complete or partial polypeptide sequence of IL-1Ra having intra-articular activity that neutralizes the effect of IL-1 on arthritis.

[0052] The helper-dependent adenovirus vectors of this disclosure preferably contain the nucleic acid sequence of IL-1Ra under the control of an inflammation-sensitive promoter. Although IL-1Ra contains a species-specific nucleic acid sequence, the adenovirus vector can express an interleukin-1 receptor antagonist (IL-1Ra) derived from any mammalian species or from humans. Preferably, the cDNA of the mammalian interleukin-1 receptor antagonist (IL-1Ra) used for cloning is a cDNA selected from the group consisting of human IL-1Ra, mouse IL-1Ra, horse IL-1Ra, dog IL-1Ra, cat IL-1Ra, rabbit IL-1Ra, hamster IL-1Ra, bovine IL-1Ra, camel IL-1Ra or their homologs in other mammalian species.

[0053] To monitor the presence of genomic vector sequences in synovial cells, the helper-dependent adenovirus vector according to this disclosure may further include sequences encoding visually or instrumentally detectable marker genes. Preferred marker genes include, but are not limited to, green fluorescent protein (GFP), LacZ, or luciferase enzymes.

[0054] As an example, the nucleic acid sequence of mouse IL-1Ra used in this disclosure is shown in the sequence listing in SEQ ID NO: 1. As stated above, any nucleic acid sequence resulting in a bioactive IL-1Ra protein of any mammal or human species can be used in the context of this disclosure. Furthermore, conserved nucleic acid sequences encoding the same amino acids, polypeptides, or proteins are within the scope of this disclosure. Preferably, the helper-dependent adenovirus vector according to this disclosure contains a nucleic acid sequence of IL-1Ra (e.g., cDNA) having at least 95%, 96%, 97%, 98%, or 99% sequence homology to the nucleic acid sequence shown in SEQ ID NO: 1. This disclosure may also include a bioactive nucleic acid sequence of IL-1Ra or a fragment thereof. Thus, the helper-dependent adenovirus vector according to this disclosure may include a bioactive fragment of the nucleic acid sequence described in SEQ ID NO: 1.

[0055] As an example, the nucleic acid sequence of human IL-1Ra used in this disclosure is shown in the sequence listing in SEQ ID NO: 4. As stated above, any nucleic acid sequence that results in a human bioactive IL-1Ra protein can be used in the context of this disclosure. Furthermore, conserved nucleic acid sequences encoding the same amino acids, polypeptides, or proteins are within the scope of this disclosure. Preferably, the helper-dependent adenovirus vector according to this disclosure contains a nucleic acid sequence of IL-1Ra (e.g., cDNA) having at least 95%, 96%, 97%, 98%, or 99% sequence homology to the nucleic acid sequence shown in SEQ ID NO: 4. This disclosure may also include a bioactive nucleic acid sequence of IL-1Ra or a fragment thereof. Thus, the helper-dependent adenovirus vector according to this disclosure may include a bioactive fragment of the nucleic acid sequence described in SEQ ID NO: 4.

[0056] As an example, the nucleic acid sequence of human IL-1Ra used in this disclosure as described in SEQ ID NO: 4 can express a human IL-1Ra protein with an amino acid sequence that is at least 95% homologous to SEQ ID NO: 6. The nucleic acid sequence of human IL-1Ra used in this disclosure as described in SEQ ID NO: 4 can express a human IL-1Ra protein with an amino acid sequence that is at least 96%, 97%, 98%, or 99% homologous to SEQ ID NO: 6. The nucleic acid sequence of human IL-1Ra used in this disclosure as described in SEQ ID NO: 4 can express a human IL-1Ra protein with an amino acid sequence that is at least 99% homologous to SEQ ID NO: 6. The nucleic acid sequence of human IL-1Ra used in this disclosure as described in SEQ ID NO: 4 can express a human IL-1Ra protein with the amino acid sequence described in SEQ ID NO: 6.

[0057] In some embodiments, human IL-1Ra may have an amino acid sequence that is at least 95% to 99% homologous to the amino acid sequence of wild-type human IL-1Ra protein. In some embodiments, human IL-1Ra may have an amino acid sequence that is 95% to 99% homologous to the human IL-1Ra protein of the amino acid sequence described in SEQ ID NO: 6.

[0058] The present invention relates to an adenovirus-based biological delivery and expression system for the treatment of osteoarthritis or osteoarthritis conditions in human joints, or for the prevention of such conditions in humans identified as being at risk of developing osteoarthritis or osteoarthritis conditions, wherein the adenovirus-based biological delivery and expression system comprises a helper-dependent adenovirus vector genome copy (GC) including a nucleic acid sequence encoding human interleukin-1 receptor antagonist (IL-1Ra), left and right reverse terminal repeats, an adenovirus packaging signal, and non-viral and non-coding stuffer nucleic acid sequences, wherein the expression of the human IL-1Ra gene encodes human IL-1Ra. The nucleic acid sequence of the adenovirus-based biological delivery and expression system, which is regulated by an NF-κB-inducible promoter located upstream of the nucleic acid sequence reading frame and includes the promoter, the nucleic acid sequence encoding IL-1Ra, left and right reverse terminal repeats, adenovirus packaging signals and non-viral, non-coding stuffer nucleic acid sequences, may be at least 95% homologous to the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7, and the adenovirus-based biological delivery and expression system is isolated from a helper-dependent adenovirus vector and host cells infected with the helper virus, and the adenovirus-based biological delivery and expression system is a) 1.4 × 10⁶ per milliliter 8 ~1.4×10 12 a) Helper-dependent adenovirus vector of GC (GC / ml); b) less than 15% helper virus particles; c) less than 10% empty capsid; d) host cell protein less than 100 μg / ml; e) host cell nucleic acid less than 20 ng / ml; f) endotoxin less than 35 EU / ml; and g) 300 GC / TCID 50 This provides an adenovirus-based biological delivery and expression system, including the following ratios of viral particles to infectious units.

[0059] The level of helper virus in the adenovirus-based biological delivery and expression systems disclosed herein may be any one of the following: less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. The level of helper virus in the adenovirus-based biological delivery and expression systems disclosed herein may be 1% to 2% of helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression systems disclosed herein may be 2% to 3% of helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression systems disclosed herein may be 3% to 4% of helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression systems disclosed herein may be 4% to 5% of helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression system disclosed herein may be 5% to 6% helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression system disclosed herein may be 6% to 7% helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression system disclosed herein may be 7% to 8% helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression system disclosed herein may be 8% to 9% helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression system disclosed herein may be 9% to 10% helper virus particles.

[0060] The level of helper virus in the adenovirus-based biological delivery and expression system disclosed herein may be 10% to 11% helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression system disclosed herein may be 11% to 12% helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression system disclosed herein may be 12% to 13% helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression system disclosed herein may be 13% to 14% helper virus particles. The level of helper virus in the adenovirus-based biological delivery and expression system disclosed herein may be less than 14% to 15% helper virus particles.

[0061] The level of empty capsids in the adenovirus-based biological delivery and expression systems disclosed herein may be any one of the following: less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. The level of empty capsids in the adenovirus-based biological delivery and expression systems disclosed herein may be 1% to 2%. The level of empty capsids in the adenovirus-based biological delivery and expression systems disclosed herein may be 2% to 3%. The level of empty capsids in the adenovirus-based biological delivery and expression systems disclosed herein may be 3% to 4%. The level of empty capsids in the adenovirus-based biological delivery and expression systems disclosed herein may be 4% to 5%. The level of empty capsids in the adenovirus-based biological delivery and expression systems disclosed herein may be 5% to 6% empty capsids. The level of empty capsids in the adenovirus-based biological delivery and expression systems disclosed herein may be 6% to 7% empty capsids. The level of empty capsids in the adenovirus-based biological delivery and expression systems disclosed herein may be 7% to 8% empty capsids. The level of empty capsids in the adenovirus-based biological delivery and expression systems disclosed herein may be 8% to 9% empty capsids. The level of empty capsids in the adenovirus-based biological delivery and expression systems disclosed herein may be 9% to less than 10% empty capsids. The levels of empty capsids and helper viruses in the adenovirus-based biological delivery and expression systems disclosed herein may be the same.

[0062] In some embodiments, the terms “empty capsid” and “empty particle” refer to an adenovirus vector virion that includes a helper-dependent adenovirus protein shell but lacks all or part of a polynucleotide construct comprising a nucleic acid encoding a human interleukin-1 receptor antagonist (IL-1Ra), left and right reverse-terminal repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence, wherein the expression of the human IL-1Ra gene is regulated by an NF-κB-inducible promoter located upstream of the leading frame of the nucleic acid sequence encoding the human IL-1Ra, and is specifically activated by a factor including, but not limited to, an immunostimulant, and the nucleic acid sequence of the adenovirus-based biological delivery and expression system comprising the promoter, the nucleic acid sequence encoding IL-1Ra, left and right reverse-terminal repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence may be at least 95% homologous to the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7.

[0063] The term “host cell” refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can be used or have been used as recipients of the helper-dependent adenovirus vector construct and helper virus of the present invention. This term includes the offspring of the transfected original cell. Thus, as used herein, “host cell” generally refers to a cell that has been transfected with an exogenous DNA sequence. Offspring of a single parent cell may not necessarily be completely identical to the original parent in morphology or in genomics or total DNA complement due to natural, accidental, or intentional mutations.

[0064] The adenovirus-based biological delivery and expression system of the present invention may have a pH of 7.0 ± 1.0. The adenovirus-based biological delivery and expression system of the present invention may have a pH of 6.0 to 6.5. The adenovirus-based biological delivery and expression system of the present invention may have a pH of 6.5 to 7.0. The adenovirus-based biological delivery and expression system of the present invention may have a pH of 7.0 to 7.5. The adenovirus-based biological delivery and expression system of the present invention may have a pH of 7.0 to 8.0.

[0065] The adenovirus-based biological delivery and expression system of the present invention may contain an osmolality of 600 mOsm / kg or less. The adenovirus-based biological delivery and expression system of the present invention may contain one osmolality of any of 100 mOsm / kg to 200 mOsm / kg. The adenovirus-based biological delivery and expression system of the present invention may contain one osmolality of any of 200 mOsm / kg to 300 mOsm / kg. The adenovirus-based biological delivery and expression system of the present invention may contain one osmolality of any of 300 mOsm / kg to 400 mOsm / kg. The adenovirus-based biological delivery and expression system of the present invention may contain one osmolality of any of 400 mOsm / kg to 500 mOsm / kg. The adenovirus-based biological delivery and expression system of the present invention may contain one osmolality of any of 500 mOsm / kg to 600 mOsm / kg.

[0066] The biological delivery and expression system based on adenovirus of the present invention is a) 1.4 × 10 9 ~1.4×10 12 b) 1.4 × 10 9 ~1.4×10 11 ; or c) 1.4 × 10 9 ~1.4×10 10 It may also contain a GC / ml helper-dependent adenovirus vector (HDAd).

[0067] The biological delivery and expression system based on adenovirus of the present invention is 1.4 × 10⁻⁶9 GC / ml or higher: 5.6 × 10 9 The invention may include a helper-dependent adenovirus vector (HDAd) with a GC / ml concentration of less than 2.8 × 10⁻¹⁶. 9 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 1.4 × 10⁻⁶ 10 GC / ml or higher: 5.6 × 10 10 The invention may include a helper-dependent adenovirus vector (HDAd) with a GC / ml concentration of less than 2.8 × 10⁻¹⁶. 10 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 1.4 × 10⁻⁶ 11 GC / ml or higher: 5.6 × 10 11 The invention may include a helper-dependent adenovirus vector (HDAd) with a GC / ml concentration of less than 2.8 × 10⁻¹⁶. 11 It may also contain a GC / ml helper-dependent adenovirus vector (HDAd).

[0068] The biological delivery and expression system based on adenovirus of the present invention is 1.4 × 10⁻⁶ 9 ~5.6×10 9 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 1.4 × 10⁻⁶ 10 ~5.6×10 10 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 1.4 × 10⁻⁶ 11 ~5.6×10 11 It may also contain a GC / ml helper-dependent adenovirus vector (HDAd).

[0069] The biological delivery and expression system based on adenovirus of the present invention is 2 × 109 ~5.6×10 9 The invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2 × 10⁻¹⁶ 10 ~5.6×10 10 The system may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system is 2 × 10⁻¹⁶. 11 ~5.6×10 11 It may contain GC / ml.

[0070] The biological delivery and expression system based on adenovirus of the present invention is 2.8 × 10⁻⁶. 9 ~5.6×10 9 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2.8 × 10⁻⁶ 10 ~5.6×10 10 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2.8 × 10⁻⁶ 11 ~5.6×10 11 It may also contain a GC / ml helper-dependent adenovirus vector (HDAd).

[0071] The biological delivery and expression system based on adenovirus of the present invention is 2 × 10 9 ~2.8×10 9 The invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2 × 10⁻¹⁶ 10 ~2.8×10 10 The invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2 × 10⁻¹⁶ 11 ~2.8×10 11 It may also contain a GC / ml helper-dependent adenovirus vector (HDAd).

[0072] The biological delivery and expression system based on adenovirus of the present invention is 1.4 × 10⁻⁶ 9 ~2.8×10 10 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 1.4 × 10⁻⁶ 10 ~2.8×10 11 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 1.4 × 10⁻⁶ 11 ~2.8×10 11 It may also contain a GC / ml helper-dependent adenovirus vector (HDAd).

[0073] The biological delivery and expression system based on adenovirus of the present invention is 2.8 × 10⁻⁶. 9 ~1.4×10 12 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2.8 × 10⁻⁶ 10 ~1.4×10 12 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2.8 × 10⁻⁶ 11 ~1.4×10 12 It may also contain a GC / ml helper-dependent adenovirus vector (HDAd).

[0074] The biological delivery and expression system based on adenovirus of the present invention is 2.8 × 10⁻⁶. 9 ~2.8×10 11 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2.8 × 10⁻⁶ 9 ~1.4×10 10 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2.8 × 10⁻⁶ 10 ~2.8×10 11It may contain helper-dependent adenovirus vectors (HDAd) of 1.4×10 GC / ml.

[0075] Adenovirus-based biological delivery and expression systems may contain 1.4×10 9 GC / ml. The adenovirus-based biological delivery and expression systems of the present invention may contain 1.4×10 10 GC / ml of helper-dependent adenovirus vectors (HDAd). The adenovirus-based biological delivery and expression systems of the present invention may contain 1.4×10 11 GC / ml of helper-dependent adenovirus vectors (HDAd). Adenovirus-based biological delivery and expression systems may contain 1.4×10 12 GC / ml.

[0076] The adenovirus-based biological delivery and expression systems of the present invention may contain 2×10 9 GC / ml of helper-dependent adenovirus vectors (HDAd). The adenovirus-based biological delivery and expression systems of the present invention may contain 2×10 10 GC / ml of helper-dependent adenovirus vectors (HDAd). The adenovirus-based biological delivery and expression systems of the present invention may contain 2×10 11 GC / ml of helper-dependent adenovirus vectors (HDAd).

[0077] The adenovirus-based biological delivery and expression systems of the present invention may contain 2.8×10 9 GC / ml of helper-dependent adenovirus vectors (HDAd). The adenovirus-based biological delivery and expression systems of the present invention may contain 2.8×10 10 GC / ml of helper-dependent adenovirus vectors (HDAd). The adenovirus-based biological delivery and expression systems of the present invention may contain 2.8×10 11 GC / ml of helper-dependent adenovirus vectors (HDAd).

[0078] The biological delivery and expression system based on adenovirus of the present invention is 5.6 × 10 9 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 5.6 × 10⁻⁶ 10 The present invention may include a GC / ml helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 5.6 × 10⁻⁶ 11 It may also contain a GC / ml helper-dependent adenovirus vector (HDAd).

[0079] The adenovirus-based biological delivery and expression system may contain a dose volume of 1 ml to 5 ml. The adenovirus-based biological delivery and expression system may contain a dose volume of 2 ml to 5 ml. The adenovirus-based biological delivery and expression system may contain a dose volume of 3 ml to 5 ml. The adenovirus-based biological delivery and expression system may contain a dose volume of 4 ml to 5 ml. The adenovirus-based biological delivery and expression system may contain a dose volume of 5 ml.

[0080] The biological delivery and expression system based on adenovirus of the present invention is 7 × 10 9 ~7×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 9 ~7×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 9 ~7×10 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0081] The biological delivery and expression system based on adenovirus of the present invention is 7 × 10 9 ~2.8×10 10The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 10 ~2.8×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 11 ~2.8×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0082] The biological delivery and expression system based on adenovirus of the present invention is 7 × 10 10 ~7×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 10 ~7×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 11 ~7×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0083] The biological delivery and expression system based on adenovirus of the present invention is 7 × 10 9 ~2.8×10 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 10 ~2.8×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 11 ~2.8×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0084] The biological delivery and expression system based on adenovirus of the present invention is 10 10 ~2.8×10 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 10 11 ~2.8×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 10 12 ~2.8×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0085] The biological delivery and expression system based on adenovirus of the present invention is 2.8 × 10⁻⁶. 9 ~5.6×10 9 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2.8 × 10⁻⁶ 10 ~5.6×10 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2.8 × 10⁻⁶. 11 ~5.6×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0086] The biological delivery and expression system based on adenovirus of the present invention is 10 10 ~1.4×10 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 10 11 ~1.4×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 10 12 ~1.4×10 12The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0087] The biological delivery and expression system based on adenovirus of the present invention is 7 × 10 9 ~5.6×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 10 ~5.6×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 11 ~5.6×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0088] The biological delivery and expression system based on adenovirus of the present invention is 1.4 × 10⁻⁶ 10 ~7×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention may contain 1.4 × 10⁻⁶ units. 11 ~7×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention may contain 1.4 × 10⁻⁶ units. 12 ~7×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0089] The biological delivery and expression system based on adenovirus of the present invention is 1.4 × 10⁻⁶ 10 ~1.4×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention may contain 1.4 × 10⁻⁶ units. 10 ~1.4×10 11The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention may contain 1.4 × 10⁻⁶ units. 11 ~1.4×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0090] The biological delivery and expression system based on adenovirus of the present invention is 7 × 10 9 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 7 × 10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0091] The biological delivery and expression system based on adenovirus of the present invention is 1.4 × 10⁻⁶ 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention may contain 1.4 × 10⁻⁶ units. 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention may contain 1.4 × 10⁻⁶ units. 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0092] The biological delivery and expression system based on adenovirus of the present invention is 2.8 × 10⁻⁶. 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2.8 × 10⁻⁶. 11The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system of the present invention is 2.8 × 10⁻⁶. 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0093] Pharmaceutical composition The adenovirus-based biological delivery and expression system of the present invention can be incorporated into a pharmaceutical composition suitable for administration. The composition of the present disclosure includes a pharmaceutical composition comprising an adenovirus helper-dependent adenovirus vector comprising a nucleic acid sequence encoding a human or mammalian interleukin-1 receptor antagonist (IL-1Ra), L ITR, R ITR, a packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence, wherein the expression of the human or mammalian interleukin-1 receptor antagonist (IL-1Ra) gene is regulated by an inflammation-sensitive promoter located upstream of the reading frame of the nucleic acid sequence encoding the human or mammalian IL-1Ra. In some embodiments, the pharmaceutical composition can be used for the treatment or prevention of osteoarthritis.

[0094] Preferred inflammation-sensitive promoters as used in the context of this disclosure are promoters that can be induced by NF-κB, interleukin-6 (Il-6), interleukin-1 (IL-1), tumor necrosis factor (TNF), cyclooxygenase-2 (COX-2), complement factor-3 (C3), serum amyloid A3 (SAA3), macrophage inflammatory protein-1a (MIP-1a), or the hybrid constructs described above. In a preferred embodiment, the inflammation-sensitive promoter is the NF-κB5-ELAM promoter.

[0095] Such compositions typically comprise the helper-dependent adenoviral vector virus particles, helper adenovirus, and a pharmaceutically acceptable carrier disclosed herein. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington’s Pharmaceutical Sciences, a standard reference text in the art, which is hereby incorporated by reference herein. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous media, such as fixed oils, can also be used. The use of such media and agents for pharmaceutical active substances is well known in the art. The use thereof in the compositions is contemplated, except in cases where any conventional media or agent is incompatible with the active compound. Additionally, auxiliary active compounds can be incorporated into the compositions.

[0096] The pharmaceutical compositions of the present invention are formulated to suit their intended route of administration. Examples of routes of administration include parenteral administration, such as intra-articular, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates; and agents for adjusting isotonicity, such as sodium chloride or dextrose. The pH can be adjusted using an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0097] Suitable pharmaceutical compositions for injectable use include sterile aqueous solutions (if water-soluble), dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (trademark) (BASF; Parsippany, N.J.), or phosphate-buffered physiological saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. For example, the use of coatings such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants can maintain appropriate fluidity. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include in the composition isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, etc., and sodium chloride. The long-term absorption of injectable compositions can be achieved by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.

[0098] If necessary, a sterile injectable solution can be prepared by incorporating the required amount of the active compound in a suitable solvent, with one or a combination of the ingredients listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile medium containing a basic dispersion medium and the other necessary ingredients derived from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying to obtain a powder of the active ingredient and further desired ingredients derived from its pre-sterilized filtered solution.

[0099] Chemical properties of adenovirus expression and delivery systems disclosed herein The capsid of the adenovirus-based biological delivery and expression system (FX201) of this disclosure may be non-enveloped and may contain 29.3 kb of double-stranded DNA. The theoretical molecular weight of the capsid may be 103.9 megadaltons (MDa), and the genome may be 18.1 MDa. The capsid of FX201 may have a diameter of approximately 100 nm.

[0100] Formulation: FX201 can be formulated in a buffer consisting of approximately 1-20 mM TRIS, approximately 50-100 mM NaCl, 0.01-1% w / v polysorbate 80, 1-10% w / v sucrose, 0.1-10 mM MgCl2, 50-500 μM EDTA, 1-5% v / v ethanol, and 5-50 mM L-histidine. In a preferred embodiment, FX201 can be formulated in a buffer comprising 10 mM TRIS, 75 mM NaCl, 0.02% w / v polysorbate 80, 5% w / v sucrose, 1.0 mM MgCl2, 100 μM EDTA, 0.5% v / v ethanol, and 10 mM L-histidine. The product may be a clear to slightly milky, colorless suspension free of visible particles.

[0101] Storage conditions and stability: FX201 can be stored as a frozen solution at -65°C or below. When stored at -65°C or below, FX201 may be stable for at least 3 months, at least 6 months, or at least 12 months. Once thawed, the product should be stored at 2-8°C and used within 7 days. FX201 can be kept at room temperature (RT) for some time. Once the vial is ready for use, it can be kept at RT for up to 7 hours (a vial kept at RT cannot be returned to refrigeration for later use). Once a dose of FX201 has been prepared in a syringe, it should be kept at RT and used within 4 hours.

[0102] The adenovirus-based biological delivery and expression system comprises a genome copy (GC) of a helper-dependent adenovirus vector containing a nucleic acid sequence encoding a human interleukin-1 receptor antagonist (IL-1Ra), left and right reverse end repeats, an adenovirus packaging signal, and non-viral and non-coding stuffer nucleic acid sequences, wherein the expression of the human IL-1Ra gene is regulated by an inflammation-sensitive promoter located upstream of the reading frame of the nucleic acid sequence encoding the human IL-1Ra, and the nucleic acid sequence of the adenovirus-based biological delivery and expression system, comprising the promoter, the nucleic acid sequence encoding IL-1Ra, left and right reverse end repeats, an adenovirus packaging signal, and non-viral and non-coding stuffer nucleic acid sequences, may be at least 95% homologous to the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7, and the adenovirus-based biological delivery and expression system contains 1.4 × 10⁶ per milliliter (ml). 8 ~1.4×10 12 Pharmaceutical compositions of the present disclosure, comprising an adenovirus-based biological delivery and expression system including a GC helper-dependent adenovirus vector, can be used for the treatment of osteoarthritis or osteoarthritis conditions in human joints, or for the prevention of such conditions in humans identified as being at risk of developing osteoarthritis or osteoarthritis conditions. The inflammation-sensitive promoter may be a promoter that can be induced by any one of NF-κB, interleukin-6 (Il-6), interleukin-1 (IL-1), tumor necrosis factor (TNF), cyclooxygenase-2 (COX-2), complement factor-3 (C3), serum amyloid A3 (SAA3), macrophage inflammatory protein-1a (MIP-1a), or a hybrid construct of the above. In a preferred embodiment, the inflammation-sensitive promoter is an NF-κB-inducible promoter. In a preferred embodiment, the NF-κB-inducible promoter is an NF-KB5-ELAM promoter.

[0103] The nucleic acid sequence of an adenovirus-based biological delivery and expression system, including the promoter, the nucleic acid sequence encoding IL-1Ra, left and right reverse end repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence, may be at least 96%, 97%, 98%, or 99% homologous to the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7. The nucleic acid sequence of an adenovirus-based biological delivery and expression system, including the promoter, the nucleic acid sequence encoding IL-1Ra, left and right reverse end repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence, may be at least 99% homologous to the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7. The nucleic acid sequence of an adenovirus-based biological delivery and expression system, including the promoter, the nucleic acid sequence encoding IL-1Ra, left and right reverse end repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence, may be that of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7.

[0104] In some embodiments, the nucleic acid sequence of the adenovirus-based biological delivery and expression system, including the promoter, the nucleic acid sequence encoding IL-1Ra, left and right reverse-ended repeats, the adenovirus packaging signal, and the non-viral, non-coding stuffer nucleic acid sequence, may be at least 95% homologous to the nucleic acid sequence of SEQ ID NO: 7.

[0105] The nucleic acid sequence encoding IL-1Ra may include the nucleic acid of SEQ ID NO: 1. The nucleic acid sequence encoding IL-1Ra may also include the nucleic acid of SEQ ID NO: 4. SEQ ID NO: 4 is a codon-optimized version of the original coding sequence of human IL-1Ra (SEQ ID NO: 5), and the codon-optimized sequence described in SEQ ID NO: 4 is a) Compared to the codon adaptation index (CAI) of 0.78 in wild-type human IL-1Ra protein, it has a CAI of 0.96. b) Compared to 56% of the codons with the highest frequency of use in wild-type human IL-1Ra protein, 85% of the codons with the highest frequency of use are c) Compared to the average GC content of 51.98 in wild-type human IL-1Ra protein, it has an average GC content of 60.4, and d) Compared to wild-type human IL-1Ra protein, it lacks negative cis elements including splice sites (GGTAAG), splice sites (GGTGAT), poly(AATAAA), poly(ATTAAA), destabilization (ATTTA), poly(TTTTTT), and poly(AAAAAAA).

[0106] The amino acid sequence of human IL-1Ra is shown in SEQ ID NO: 6.

[0107] The nucleic acid sequence of the adenovirus-based biological delivery and expression system, comprising a promoter, a nucleic acid sequence encoding IL-1Ra, left and right reverse terminal repeats, an adenovirus packaging signal, and a non-viral, non-coding stuffer nucleic acid sequence, may, essentially be, or be derived from the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7.

[0108] The helper-dependent adenovirus vector may further include a marker gene encoding a protein product that is visually or instrumentally detectable to monitor the presence of the vector sequence in infected cells. The marker gene may encode one of the following: a fluorescent protein, an enzyme, or a detectable cell surface protein. The marker gene may encode one of the following: green fluorescent protein, LacZ, or the luciferase enzyme.

[0109] The pharmaceutical compositions of this disclosure, including biological delivery and expression systems based on adenoviruses, contain a) 1.4 × 10 per 1 ml of the pharmaceutical composition.9 ~1.4×10 12 b) 1.4 × 10 9 ~1.4×10 11 ; or c) 1.4 × 10 9 ~1.4×10 10 It may also include a genome copy (GC) helper-dependent adenovirus vector (HDAd).

[0110] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, contain 1.4 × 10 per ml of pharmaceutical composition. 10 ~1.4×10 12 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 1.4 × 10⁶ per ml of the pharmaceutical composition. 10 ~1.4×10 11 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 1.4 × 10⁶ per ml of the pharmaceutical composition. 11 ~1.4×10 12 It may also include a genome copy (GC) helper-dependent adenovirus vector (HDAd).

[0111] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, contain 1.4 × 10 per ml of pharmaceutical composition. 9 ~5.6×10 9 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 1.4 × 10⁶ per ml of the pharmaceutical composition. 10 ~5.6×10 10 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 1.4 × 10⁶ per ml of the pharmaceutical composition. 11 ~5.6×10 11It may also include a genome copy (GC) helper-dependent adenovirus vector (HDAd).

[0112] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, contain 2 × 10⁶ particles per 1 ml of the pharmaceutical composition. 9 ~5.6×10 9 The pharmaceutical composition of this disclosure, which includes an adenovirus-based biological delivery and expression system, may contain 2 × 10⁶ molecules per 1 ml of the pharmaceutical composition. 10 ~5.6×10 10 The pharmaceutical composition of this disclosure, which includes an adenovirus-based biological delivery and expression system, may contain 2 × 10⁶ molecules per 1 ml of the pharmaceutical composition. 11 ~5.6×10 11 It may also include a genome copy (GC) helper-dependent adenovirus vector (HDAd).

[0113] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, contain 2.8 × 10⁶ particles per 1 ml of the pharmaceutical composition. 9 ~5.6×10 9 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 2.8 × 10⁶ particles per ml of the pharmaceutical composition. 10 ~5.6×10 10 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 2.8 × 10⁶ particles per ml of the pharmaceutical composition. 11 ~5.6×10 11 It may also include a genome copy (GC) helper-dependent adenovirus vector (HDAd).

[0114] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, contain 2 × 10⁶ particles per 1 ml of the pharmaceutical composition.9 ~2.8×10 9 The pharmaceutical composition of this disclosure, which includes an adenovirus-based biological delivery and expression system, may contain 2 × 10⁶ molecules per 1 ml of the pharmaceutical composition. 10 ~2.8×10 10 The pharmaceutical composition of this disclosure, which includes an adenovirus-based biological delivery and expression system, may contain 2 × 10⁶ molecules per 1 ml of the pharmaceutical composition. 11 ~2.8×10 11 It may also include a genome copy (GC) helper-dependent adenovirus vector (HDAd).

[0115] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, contain 1.4 × 10 per ml of pharmaceutical composition. 9 ~2.8×10 10 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 1.4 × 10⁶ per ml of the pharmaceutical composition. 10 ~2.8×10 11 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 1.4 × 10⁶ per ml of the pharmaceutical composition. 11 ~2.8×10 11 It may also include a genome copy (GC) helper-dependent adenovirus vector (HDAd).

[0116] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, contain 2.8 × 10⁶ particles per 1 ml of the pharmaceutical composition. 9 ~1.4×10 12 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 2.8 × 10⁶ particles per ml of the pharmaceutical composition. 10~1.4×10 12 It may contain a helper-dependent adenovirus vector (HDAd) with a genomic copy (GC). The pharmaceutical composition of the present disclosure containing a biological delivery and expression system based on adenovirus contains, per 1 ml of the pharmaceutical composition, 2.8×10 11 ~1.4×10 12 It may contain a helper-dependent adenovirus vector (HDAd) with a genomic copy (GC).

[0117] [[]]The pharmaceutical composition of the present disclosure containing a biological delivery and expression system based on adenovirus contains, per 1 ml of the pharmaceutical composition, 2.8×10 9 ~2.8×10 11 It may contain a helper-dependent adenovirus vector (HDAd) with a genomic copy (GC). The pharmaceutical composition of the present disclosure containing a biological delivery and expression system based on adenovirus contains, per 1 ml of the pharmaceutical composition, 2.8×10 9 ~1.4×10 10 It may contain a helper-dependent adenovirus vector (HDAd) with a genomic copy (GC). The pharmaceutical composition of the present disclosure containing a biological delivery and expression system based on adenovirus contains, per 1 ml of the pharmaceutical composition, 2.8×A0 10 ~2.8×10 11 It may contain a helper-dependent adenovirus vector (HDAd) with a genomic copy (GC).

[0118] The pharmaceutical composition of the present disclosure containing a biological delivery and expression system based on adenovirus contains, per 1 ml of the pharmaceutical composition, 1.4×10 9 It may contain a helper-dependent adenovirus vector (HDAd) with a genomic copy (GC). The pharmaceutical composition of the present disclosure containing a biological delivery and expression system based on adenovirus contains, per 1 ml of the pharmaceutical composition, 1.4×10 10 It may contain a helper-dependent adenovirus vector (HDAd) with a genomic copy (GC). The pharmaceutical composition of the present disclosure containing a biological delivery and expression system based on adenovirus contains, per 1 ml of the pharmaceutical composition, 1.4×10 11The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 1.4 × 10⁶ per ml of the pharmaceutical composition. 12 It may also include a genome copy (GC) helper-dependent adenovirus vector (HDAd).

[0119] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, contain 2 × 10⁶ particles per 1 ml of the pharmaceutical composition. 9 The pharmaceutical composition of this disclosure, which includes an adenovirus-based biological delivery and expression system, may contain 2 × 10⁶ molecules per 1 ml of the pharmaceutical composition. 10 The pharmaceutical composition of this disclosure, which includes an adenovirus-based biological delivery and expression system, may contain 2 × 10⁶ molecules per 1 ml of the pharmaceutical composition. 11 It may also include a genome copy (GC) helper-dependent adenovirus vector (HDAd).

[0120] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, contain 2.8 × 10⁶ particles per 1 ml of the pharmaceutical composition. 9 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 2.8 × 10⁶ particles per ml of the pharmaceutical composition. 10 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 2.8 × 10⁶ particles per ml of the pharmaceutical composition. 11 It may also include a genome copy (GC) helper-dependent adenovirus vector (HDAd).

[0121] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, contain 5.6 × 10 per ml of pharmaceutical composition. 9The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 5.6 × 10⁶ molecules per ml of the pharmaceutical composition. 10 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain 5.6 × 10⁶ molecules per ml of the pharmaceutical composition. 11 It may also include a genome copy (GC) helper-dependent adenovirus vector (HDAd).

[0122] The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a dose volume of 1 ml to 5 ml. The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a dose volume of 2 ml to 5 ml. The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a dose volume of 4 ml to 5 ml. The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a dose volume of 3 ml to 5 ml. The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a dose volume up to 5 ml.

[0123] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, are 7 × 10 9 ~7×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 7 × 10 9 ~7×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 7 × 10 9 ~7×10 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0124] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, are 7 × 10 10 ~7×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 7 × 10 10 ~7×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 7 × 10 11 ~7×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0125] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, are 7 × 10 9 ~2.8×10 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 7 × 10 10 ~2.8×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 7 × 10 11 ~2.8×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0126] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, are 7 × 10 9 ~2.8×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 7 × 10 9 ~2.8×10 12The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 7 × 10 10 ~2.8×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0127] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, are 1 × 10 10 ~2.8×10 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 1 × 10⁻⁶. 11 ~2.8×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system is 1 × 10⁻⁶ 12 ~2.8×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0128] The pharmaceutical compositions of this disclosure, including biological delivery and expression systems based on adenoviruses, are 2.8 × 10 9 ~5.6×10 9 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a total dose of GC-dependent helper adenovirus vector (HDAd). 10 ~5.6×10 10 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a total dose of GC-dependent helper adenovirus vector (HDAd). 11 ~5.6×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0129] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, are 1 × 10 10~1.4×10 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 1 × 10⁻⁶. 11 ~1.4×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 1 × 10⁻⁶. 12 ~1.4×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0130] Adenovirus-based biological delivery and expression systems are 7 × 10 9 ~5.6×10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system is 7 × 10⁻⁶. 10 ~5.6×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The adenovirus-based biological delivery and expression system is 7 × 10⁻⁶. 11 ~5.6×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0131] The pharmaceutical compositions of this disclosure, including biological delivery and expression systems based on adenoviruses, are 1.4 × 10 10 ~7×10 12 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a total dose of GC-dependent helper adenovirus vector (HDAd). 11 ~7×10 12 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a total dose of GC-dependent helper adenovirus vector (HDAd). 12 ~7×10 12The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0132] The pharmaceutical compositions of this disclosure, including biological delivery and expression systems based on adenoviruses, are 1.4 × 10 10 ~1.4×10 12 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a total dose of GC-dependent helper adenovirus vector (HDAd). 10 ~1.4×10 11 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a total dose of GC-dependent helper adenovirus vector (HDAd). 11 ~1.4×10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0133] The pharmaceutical compositions of this disclosure, including a biological delivery and expression system based on adenovirus, are 7 × 10 9 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 7 × 10 10 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 7 × 10 11 The total dose of GC may include a helper-dependent adenovirus vector (HDAd). The pharmaceutical compositions of this disclosure, including an adenovirus-based biological delivery and expression system, may be 7 × 10 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0134] The pharmaceutical compositions of this disclosure, including biological delivery and expression systems based on adenoviruses, are 1.4 × 10 10The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a total dose of GC-dependent helper adenovirus vector (HDAd). 11 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a total dose of GC-dependent helper adenovirus vector (HDAd). 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0135] The pharmaceutical compositions of this disclosure, including biological delivery and expression systems based on adenoviruses, are 2.8 × 10 10 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a total dose of GC-dependent helper adenovirus vector (HDAd). 11 The pharmaceutical composition of this disclosure, including an adenovirus-based biological delivery and expression system, may contain a total dose of GC-dependent helper adenovirus vector (HDAd). 12 The total dose of GC may include a helper-dependent adenovirus vector (HDAd).

[0136] The pharmaceutical compositions of this disclosure can be formulated for intratendinous, intramuscular, intraarticular, or subacromial injection into human joints. The pharmaceutical compositions are formulated for intraarticular injection into human joints.

[0137] The pharmaceutical composition comprising the adenovirus-based biological delivery and expression system of the present invention may contain viral particles of a helper-dependent adenovirus vector quantified as the genome copy (GC) of the helper-dependent adenovirus vector per milliliter (ml), or viral particles of the helper-dependent adenovirus vector (VP) per milliliter (ml), where 1 VP / ml corresponds to 1.4 GC / ml.

[0138] The pharmaceutical composition of the present invention contains 1.4 × 10 per milliliter (ml). 8 ~1.4×1012 The pharmaceutical composition may also include an adenovirus-based biological delivery and expression system containing a genome copy helper-dependent adenovirus vector (GC), but the composition may also contain 10 per milliliter (ml) of synovial fluid in the joint. 8 ~10 12 The present disclosure may include a helper-dependent adenovirus vector of viral particles (VP).

[0139] In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system is 10 per 1 ml of synovial fluid in the joint. 9 ~10 12 ;10 9 ~10 11 ; or 10 9 ~10 10 The VP may contain a helper-dependent adenovirus vector. Preferably, the pharmaceutical composition comprising an adenovirus-based biological delivery and expression system is present in a concentration of 10 per 1 ml of synovial fluid in the joint. 9 ~10 11 It may also contain a helper-dependent adenovirus vector for VP.

[0140] In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system delivers 2.8 × 10⁶ units per 1 ml of synovial fluid in the joint. 9 ~2.8×10 11 The invention may also include a helper-dependent adenovirus vector of VP. In another aspect of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system may contain 2.8 × 10⁶ per ml of synovial fluid in the joint. 9 ~2.8×10 10 The invention may also include a helper-dependent adenovirus vector of VP. In certain embodiments of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system delivers 2.8 × 10⁶ per ml of synovial fluid in the joint. 10 ~2.8×10 11 It may also contain a helper-dependent adenovirus vector for VP.

[0141] In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system is 2 × 10 per 1 ml of synovial fluid in the joint. 9 ~2×10 11 The VP may contain a helper-dependent adenovirus vector. In another aspect of the present invention, the adenovirus-based biological delivery and expression system is 2 × 10⁶ per 1 ml of synovial fluid in the joint. 9 ~2×10 10 The invention may include a helper-dependent adenovirus vector of VP. In certain embodiments of the present invention, the pharmaceutical composition comprising an adenovirus-based biological delivery and expression system contains 2 × 10⁶ per 1 ml of synovial fluid in the joint. 10 ~2×10 11 It may also contain a helper-dependent adenovirus vector for VP.

[0142] In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system delivers 2.8 × 10⁶ units per 1 ml of synovial fluid in the joint. 9 The invention may also include a helper-dependent adenovirus vector of VP. In another aspect of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system may contain 2.8 × 10⁶ per ml of synovial fluid in the joint. 10 The invention may also include a helper-dependent adenovirus vector of VP. In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system delivers 2.8 × 10⁶ per ml of synovial fluid in the joint. 11 The invention may also include a helper-dependent adenovirus vector of VP. In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system delivers 2.8 × 10⁶ per ml of synovial fluid in the joint. 11 It may also contain a helper-dependent adenovirus vector for VP.

[0143] In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system is 10 per 1 ml of synovial fluid in the joint. 9 ~10 12 ;10 9 ~10 11 ; or 10 9 ~1010 The VP may contain a helper-dependent adenovirus vector. Preferably, the pharmaceutical composition comprising an adenovirus-based biological delivery and expression system is present in a concentration of 10 per 1 ml of synovial fluid in the joint. 9 ~10 11 It may also contain a helper-dependent adenovirus vector for VP.

[0144] A method for infecting articular cells of one or more osteoarthritis-affected joints of a person suffering from osteoarthritis or an osteoarthritis-affected condition with the adenovirus-based biological delivery and expression system of the present invention involves infecting one or more osteoarthritis-affected joints of a person requiring it with 10 per milliliter (ml) of synovial fluid in the joint. 8 ~10 12 This may include infecting a helper-dependent adenovirus vector of the virus particles (VP) of the present disclosure.

[0145] In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system is 10 per 1 ml of synovial fluid in the joint. 9 ~10 12 ;10 9 ~10 11 ; or 10 9 ~10 10 The VP may contain a helper-dependent adenovirus vector. Preferably, the pharmaceutical composition comprising an adenovirus-based biological delivery and expression system is present in a concentration of 10 per 1 ml of synovial fluid in the joint. 9 ~10 11 It may also contain a helper-dependent adenovirus vector for VP.

[0146] In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system delivers 2.8 × 10⁶ units per 1 ml of synovial fluid in the joint. 9 ~2.8×10 11 The invention may also include a helper-dependent adenovirus vector of VP. In another aspect of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system may contain 2.8 × 10⁶ per ml of synovial fluid in the joint. 9 ~2.8×1010 The invention may include a helper-dependent adenovirus vector of VP. In certain embodiments of the present invention, the pharmaceutical composition comprising an adenovirus-based biological delivery and expression system contains 2 × 10⁶ per 1 ml of synovial fluid in the joint. 10 ~2.8×10 11 It may also contain a helper-dependent adenovirus vector for VP.

[0147] In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system delivers 2.8 × 10⁶ units per 1 ml of synovial fluid in the joint. 9 The invention may also include a helper-dependent adenovirus vector of VP. In another aspect of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system may contain 2.8 × 10⁶ per ml of synovial fluid in the joint. 10 The invention may also include a helper-dependent adenovirus vector of VP. In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system delivers 2.8 × 10⁶ per ml of synovial fluid in the joint. 11 The invention may also include a helper-dependent adenovirus vector of VP. In some aspects of the present invention, a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system delivers 2.8 × 10⁶ per ml of synovial fluid in the joint. 11 The VP may contain a helper-dependent adenovirus vector. In some aspects of the present invention, the joint contains about 0.5 ml to about 20 ml of synovial fluid. In some aspects of the present invention, the joint may contain about 0.5 ml to 10 ml of synovial fluid. In some aspects of the present invention, the joint may contain about 0.5 ml to 5 ml of synovial fluid.

[0148] Method of Disclosure The present invention provides a method for infecting articular cells of one or more osteoarthritis-affected joints of a human suffering from osteoarthritis or an osteoarthritis-affected condition with an adenovirus-based biological delivery and expression system, comprising the steps of: a) infecting articular cells of an osteoarthritis-affected joint of a human requiring such treatment with a pharmaceutical composition comprising the adenovirus-based biological delivery and expression system disclosed herein; and b) expressing IL-1Ra in a target region within the osteoarthritis-affected joint.

[0149] Articular cells can be infected once with an adenovirus-based biological delivery and expression system. Articular cells can be infected two or more times with an adenovirus-based biological delivery and expression system.

[0150] When articular cells are infected with an adenovirus-based biological delivery and expression system more than once, each infection contains a helper-dependent adenovirus vector with a different number of genomic copies. When articular cells are infected with an adenovirus-based biological delivery and expression system at least twice, the first infection may contain a GC count per ml that is smaller than the GC count per ml of the second or any subsequent infection.

[0151] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 9 GC / ml ~ 1.4 × 10 10 GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 11 ~1.4×10 12 It may contain GC / ml.

[0152] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 10 ~1.4×10 11GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 11 ~1.4×10 12 It may contain GC / ml.

[0153] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 9 ~1.4×10 10 GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 11 ~1.4×10 11 It may contain GC / ml.

[0154] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 9 GC / ml ~ 5.6 × 10 9 GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 10 ~5.6×10 10 It may contain GC / ml.

[0155] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 10 ~5.6×10 10 GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 11 ~5.6×10 11 It may contain GC / ml.

[0156] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 9 ~5.6×10 9GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 11 ~5.6×10 11 It may contain GC / ml.

[0157] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 2.8 × 10⁶ 9 GC / ml may be included, and for the second or subsequent infection, use 2.8 × 10⁻⁶ 10 It may contain GC / ml.

[0158] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 2.8 × 10⁶ 10 GC / ml may be included, and for the second or subsequent infection, use 2.8 × 10⁻⁶ 11 It may contain GC / ml.

[0159] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 2.8 × 10⁶ 9 GC / ml may be included, and for the second or subsequent infection, use 2.8 × 10⁻⁶ 11 It may contain GC / ml.

[0160] When articular cells are infected with an adenovirus-based biological delivery and expression system at least twice, the first infection may contain a GC count per ml greater than that of the second or any subsequent infection.

[0161] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 11 ~1.4×1012 GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 9 ~1.4×10 10 It may contain GC / ml.

[0162] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 11 ~1.4×10 12 GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 10 ~1.4×10 11 It may contain GC / ml.

[0163] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 10 ~1.4×10 11 GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 9 ~1.4×10 10 It may contain GC / ml.

[0164] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 10 ~5.6×10 10 GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 9 GC / ml ~ 5.6 × 10 9 It may contain GC / ml.

[0165] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 11 ~5.6×1011 GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 10 ~5.6×10 10 It may contain GC / ml.

[0166] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 1.4 × 10⁶ 11 ~5.6×10 11 GC / ml may be included, and for the second or subsequent infection, 1.4 × 10 9 ~5.6×10 9 It may contain GC / ml.

[0167] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 2.8 × 10⁶ 10 GC / ml may be included, and for the second or subsequent infection, use 2.8 × 10⁻⁶ 9 It may contain GC / ml.

[0168] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 2.8 × 10⁶ 11 GC / ml may be included, and for the second or subsequent infection, use 2.8 × 10⁻⁶ 10 It may contain GC / ml.

[0169] When articular cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection involves a helper-dependent adenovirus vector with a different number of genome copies, and the first infection involves 2.8 × 10⁶ 11 GC / ml may be included, and for the second or subsequent infection, use 2.8 × 10⁻⁶ 9 It may contain GC / ml.

[0170] When articular cells are infected with an adenovirus-based biological delivery and expression system more than once, each infection may contain the same number of helper-dependent adenovirus vectors.

[0171] When articular cells are infected at least twice with an adenovirus-based biological delivery and expression system, each infection yields 1.4 × 10⁻⁶ 9 ~5.6×10 9 It may contain GC / ml. When articular cells are infected with an adenovirus-based biological delivery and expression system at least twice, each infection is 1.4 × 10⁻⁶ 10 ~5.6×10 10 It may contain GC / ml. When articular cells are infected with an adenovirus-based biological delivery and expression system at least twice, each infection is 1.4 × 10⁻⁶ 11 ~5.6×10 11 It may contain GC / ml.

[0172] When articular cells are infected at least twice with an adenovirus-based biological delivery and expression system, each infection yields 2.8 × 10⁻⁶ 9 It may contain GC / ml. When articular cells are infected with an adenovirus-based biological delivery and expression system at least twice, each infection is 2.8 × 10⁻⁶ 10 It may contain GC / ml. When articular cells are infected with an adenovirus-based biological delivery and expression system at least twice, each infection is 2.8 × 10⁻⁶ 11 It may contain GC / ml.

[0173] When joint cells are infected more than once with an adenovirus-based biological delivery and expression system, each infection can be performed in the same osteoarthritis-affected joint in a human.

[0174] When joint cells are infected more than once with an adenovirus-based biological delivery and expression system, all subsequent infections, including the second infection, can be performed in a different osteoarthritis-affected joint from the one in which the previous infection occurred.

[0175] Arthrocyte infection may include intratendinous, intramuscular, intraarticular, or subacromial injection of the pharmaceutical composition of this disclosure. Arthrocyte infection may include intraarticular injection of the pharmaceutical composition of this disclosure. As used herein, “arthrocyte infection” means administering the pharmaceutical composition of the present invention to a joint affected by osteoarthritis or an osteoarthritis condition, and the administration includes injecting the pharmaceutical composition into the joint affected by osteoarthritis or an osteoarthritis condition, intraarticular, intratendinous, intramuscular, or subacromial. In preferred embodiments, administration of the pharmaceutical composition of the present invention to a joint affected by osteoarthritis or an osteoarthritis condition is carried out by intraarticular injection of the pharmaceutical composition into the joint affected by osteoarthritis or an osteoarthritis condition.

[0176] Monitoring of treatment The method of the present disclosure may further include the step of (c) after the expression of IL-1Ra in a target region within the osteoarthritis-affected joint, the treatment of or monitoring of osteoarthritis or the progression of an osteoarthritis-affected joint.

[0177] Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints can be performed by determining the human pain, physical function, overall patient assessment, and joint imaging that necessitate it. Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may include assessing the progression of osteoarthritis using the Western Ontario McMasters Universities Osteoarthritis (WOMAC) index. Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may include assessing the progression of osteoarthritis using the Knee Injury and Osteoarthritis Outcome Score (KOOS). Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may include assessing the progression of osteoarthritis using the Average Daily Pain (ADP) scoring system. Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may include assessing the progression of osteoarthritis using WOMAC, KOOS, and ADP.

[0178] Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may include a physical examination of the human joint in need of treatment for any one or all of the following: joint pain, joint stiffness, friction, redness, tenderness, Baker's cysts, and joint swelling or a combination thereof. Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may also include a physical examination of the human in need of treatment for depression, sleep deprivation, hyperalgesia, central sensitization, and catastrophization or a combination thereof.

[0179] Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may include the use of radiographic imaging to determine osteophyte formation and joint space narrowing (JSN). Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may include imaging of the human joint in need using one or a combination of magnetic resonance imaging (MRI), ultrasound (US), and optical coherence tomography (OCT). Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may include the measurement of indicator interleukin-1 receptor antagonist (IL-1Ra) and interleukin-1 beta (IL-1β) protein concentrations in the knee. Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may include evaluating the immunological response to the helper-dependent adenovirus vector (HDAd) of the present invention. Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may include testing human blood samples treated with the pharmaceutical composition or method of the present invention for the presence of anti-capsid and anti-IL-1Ra antibodies. Treatment or monitoring of the progression of osteoarthritis or osteoarthritis in human joints may also include testing the concentrations of IL-1Ra and IL-1β proteins in human IA synovial fluid samples treated with the pharmaceutical composition or method of the present invention.

[0180] The method may further include (d) a step of continuing to administer an adenovirus-based biological delivery and expression system to a human joint affected by osteoarthritis if a step of monitoring the treatment or progression of osteoarthritis or an osteoarthritis condition in an affected joint indicates that osteoarthritis or an osteoarthritis condition in the human joint is not under control or treatment; or (e) a step of further adjusting the genomic copy number of the helper-dependent adenovirus vector in an adenovirus-based biological delivery and expression system and administering it to a human joint affected by osteoarthritis if a step of monitoring the treatment or progression of osteoarthritis or an osteoarthritis condition in an affected joint indicates that osteoarthritis or an osteoarthritis condition in the human joint has progressed.

[0181] Target, osteoarthritis and osteoarthritis condition In the method of this disclosure, a person suffering from osteoarthritis or an osteoarthritis-like condition may be male or female. A person suffering from osteoarthritis or an osteoarthritis-like condition may be female.

[0182] A person suffering from osteoarthritis or an osteoarthritis-like condition may be between 30 and 80 years old. A person suffering from osteoarthritis or an osteoarthritis-like condition may be over 80 years old. A person suffering from osteoarthritis or an osteoarthritis-like condition may have osteoarthritis of the joints. A person suffering from osteoarthritis or an osteoarthritis-like condition may have osteoarthritis of the shoulder, buttocks, ankle, knee, hand, or spine. A person suffering from osteoarthritis or an osteoarthritis-like condition may have knee osteoarthritis (OAK). A person suffering from osteoarthritis or an osteoarthritis-like condition may have painful knee OA that indicates a Kellgren-Lawrence (KL) grade of 2, 3, or 4. Humans may have osteoarthritis or a degenerative condition caused by aging, sex (female) related predisposition, obesity, metabolic disorders, joint injury, repetitive stress on joints, genetic predisposition, or bone deformity or a combination thereof. Humans may have osteoarthritis or a degenerative condition caused by joint injury resulting in cartilage rupture, joint dislocation, or ligament injury or a combination thereof. Humans may have osteoarthritis or a degenerative condition caused by deformation and rupture of the anterior cruciate ligament (ACL). Humans may have osteoarthritis or a degenerative condition caused by deformation and rupture of the meniscus.

[0183] Osteoarthritis is the most commonly diagnosed type of arthritis of the joints and can affect the shoulders, hands, knees, toes, fingers, wrists, and buttocks. Knee osteoarthritis is known to affect joint function, causing functional knee pain and, as it progresses, even physical disability. There are various grades of knee osteoarthritis (OA), as characterized by Kellgren & Lawrence, 1997, with 0 assigned to a normal, healthy knee, and progressing to the right, grade 4 being the most severe OA.

[0184] Grade 1 is characterized by suspected joint cavity narrowing and possible osteoproliferative marginal osteophytes. OA patients will develop very small abrasions and osteophyte growths at the distal end of the knee joint. However, at this stage, you are unlikely to experience pain or discomfort.

[0185] Grade 2 (lowest) is characterized by apparent osteophytes and the possibility of joint cavity narrowing, where diagnostic images or X-rays of the knee joint show more osteophyte proliferation, and the space between the bones appears normal, but people will begin to experience symptoms of joint pain. Typically, the area around the knee joint will feel stiff and uncomfortable, especially after getting up in the morning or after sitting for long periods of time after work. The cartilage and soft tissues retain a healthy size, but there is proteolytic breakdown of the cartilage matrix resulting from increased production of enzymes such as metalloproteinases.

[0186] Grade 3 (moderate) is characterized by moderate multiple osteophytes, obvious narrowing of the joint cavity, and the possibility of some sclerosis and epiphyseal deformation. There is obvious erosion of the cartilage surface between the bones, and fibrosis narrows the gap between the bones. As the disease progresses, proteoglycans and collagen fragments are released into the synovial fluid, and osteophytes develop in the joint as the bone becomes rougher.

[0187] Grade 4 (severe) is characterized by large osteophytes, marked narrowing of the joint cavity, severe sclerosis, and obvious epiphyseal deformities. The joint cavity between the bones is significantly reduced, causing cartilage wear and resulting in joint stiffness. Cartilage destruction leads to a chronic inflammatory response, and reduced synovial fluid causes friction, greater pain, and discomfort when walking or moving the joint. Production of synovial metalloproteinases, cytokines, and TNF increases, which can spread back to the cartilage and destroy the soft tissues around the knee. The disease progresses to a stage where more osteophytes develop, causing unbearable pain and making even everyday tasks such as walking and going down stairs difficult.

[0188] As osteoarthritis of the knee progresses, there is noticeable joint inflammation, causing frequent pain when walking, running, squatting, stretching, or kneeling. Along with joint stiffness after sitting for long periods or upon waking in the morning, there may be popping or cracking sounds when walking.

[0189] People suffering from osteoarthritis or an osteoarthritis-like condition should consume 40 kilograms per square meter (kg / m²). 2 ) may have a body mass index (BMI) below the specified range. Persons with osteoarthritis or an osteoarthritis condition may have symptoms associated with indicative knee OA for 12 months or longer. Persons with osteoarthritis or an osteoarthritis condition may have a knee pain index for more than 15 days in the last month prior to treatment with the pharmaceutical composition or method of the present invention. Persons with osteoarthritis or an osteoarthritis condition may have one or a combination of the following features as defined by the American College of Rheumatology (ACR) criteria (clinical and radiological) for OA: a) knee pain, b) (i) age over 50 years; (ii) morning stiffness of less than 30 minutes, and (iii) friction sounds when moving the knee, and c) osteophytes.

[0190] A person with osteoarthritis or an osteoarthritis-like condition may have failed two or more types of conservative therapy for the osteoarthritis index. A person with osteoarthritis or an osteoarthritis-like condition may have failed a planned athletic exercise program. A person with osteoarthritis or an osteoarthritis-like condition may have failed previous treatment with topical nonsteroidal anti-inflammatory drugs (NSAIDs). A person with osteoarthritis or an osteoarthritis-like condition may have failed previous treatment with topical nonsteroidal anti-inflammatory drugs (NSAIDs). A person with osteoarthritis or an osteoarthritis-like condition may have failed previous treatment with nonselective NSAIDs or COX-2 inhibitors. A person with osteoarthritis or an osteoarthritis-like condition may have failed one previous type of conservative therapy and at least one previous indexive knee IA treatment (corticosteroid or hyaluronic acid).

[0191] A person suffering from osteoarthritis or a degenerative condition may have a Kellgren-Lawrence (KL) grade 2 on a benchmark knee based on X-ray and physical examination. A person suffering from osteoarthritis or a degenerative condition may have a Kellgren-Lawrence (KL) grade 3 on a benchmark knee based on X-ray and physical examination. A person suffering from osteoarthritis or a degenerative condition may have a Kellgren-Lawrence (KL) grade 4 on a benchmark knee based on X-ray and physical examination.

[0192] A person suffering from osteoarthritis or an osteoarthritis-like condition may have a benchmark knee, and the area intended for injection of the pharmaceutical composition of the present invention may not show any signs of local or joint infection. A person suffering from osteoarthritis or an osteoarthritis-like condition may have a Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC®) pain score at a benchmark knee, including the endpoint, between 4.0 and 9.0 (NRS on a 0-10 scale). A person suffering from osteoarthritis or an osteoarthritis-like condition may be a non-pregnant woman or a woman who can use one or more methods of contraception at the time of treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention, or for at least 12 months thereafter.

[0193] A person with osteoarthritis or an osteoarthritis-like condition does not need to have a current or previous diagnosis of reactive arthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or arthritis associated with inflammatory bowel disease. A person with osteoarthritis or an osteoarthritis-like condition does not need to have current clinical signs and symptoms of active crystalline disease, including gout or calcium pyrophosphate deposition, in the index knee. A person with osteoarthritis or an osteoarthritis-like condition does not need to have current clinical signs and symptoms of active crystalline disease within three months prior to treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention. A person with osteoarthritis or an osteoarthritis-like condition does not need to be unable to undergo magnetic resonance imaging (MRI) due to the presence of surgical hardware or other foreign bodies in the index knee. A person with osteoarthritis or an osteoarthritis-like condition does not need to have an unstable index knee joint.

[0194] A person suffering from osteoarthritis or an osteoarthritis-like condition does not need to have received prior treatment with intra-articular (IA) drugs / biopharmaceuticals in the reference knee within six months of treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention. A person suffering from osteoarthritis or an osteoarthritis-like condition does not need to have received treatment with one or a combination of corticosteroids, hyaluronic acid, platelet-rich plasma, stem cells, prolotherapy, and amniotic fluid injection within six months of treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention. A person suffering from osteoarthritis or an osteoarthritis-like condition does not need to have undergone cryotherapy or radiofrequency nerve ablation of the reference knee within twelve months of treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention. A person suffering from osteoarthritis or an osteoarthritis-like condition does not need to have undergone arthroscopic surgery or open surgery on the reference knee within twelve months of treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention. Persons suffering from osteoarthritis or an osteoarthritis-like condition do not need to have undergone planned or anticipated surgery on the indicator knee within 12 months of treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention. Persons suffering from osteoarthritis or an osteoarthritis-like condition do not need to have loss of skin integrity on the indicator knee where the intra-articular injection is to be administered.

[0195] Individuals with osteoarthritis or an osteoarthritis-like condition do not need to show clinical test values ​​for human immunodeficiency virus (HIV) infection, a positive test for hepatitis B surface antigen (HBsAg), or a positive test for hepatitis C virus (HCV), in addition to a positive test for hepatitis C virus ribonucleic acid (HCV RNA).

[0196] Persons with osteoarthritis or an osteoarthritis-like condition do not need to show ECG abnormalities. Persons with osteoarthritis or an osteoarthritis-like condition do not need to have received or used immunomodulators, immunosuppressants, or chemotherapeutic agents within 5 years of treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention. Persons with osteoarthritis or an osteoarthritis-like condition do not need to have received any prior trial-investigation or approved gene therapy treatment within 5 years of treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention, except for excised basal cell carcinoma, squamous cell carcinoma of the skin, or effectively controlled cervical intraepithelial neoplasia, and do not have an active malignancy or a history of malignancy.

[0197] Persons suffering from osteoarthritis or an osteoarthritis-like condition may not have received active drug treatment for depression, including selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), non-selective serotonin reuptake inhibitors (NSRIs), or tricyclic antidepressants, if the dose / regimen has not been stable for at least six months prior to treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention. Persons suffering from osteoarthritis or an osteoarthritis-like condition may not have engaged in active substance abuse (drugs or alcohol) or have a history of substance abuse within 12 months prior to treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention. Persons suffering from osteoarthritis or an osteoarthritis-like condition may not have received investigational drugs, biopharmaceuticals, or devices within 3 months prior to treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention.

[0198] Persons suffering from osteoarthritis or an osteoarthritis-like condition do not have a systemic or local bacterial or viral infection requiring intravenous (IV) antibiotics or antiviral agents within four weeks prior to treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention, or oral antibiotics or antiviral agents within two weeks prior to treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention.

[0199] A person suffering from osteoarthritis or an osteoarthritis-like condition may have OA in both knees within one month prior to treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention, but the pain in the opposite knee is not 4.0 or higher (on an NRS scale of 0 to 10). A person suffering from osteoarthritis or an osteoarthritis-like condition may not have undergone total or partial knee replacement surgery on the indicator knee. A person suffering from osteoarthritis or an osteoarthritis-like condition may not have a body temperature above 99.5°F at the time of treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention.

[0200] Individuals with osteoarthritis or an osteoarthritis-like condition do not need to have a prothrombin time (PT) / international normalized ratio (INR) greater than 1.5. Individuals with osteoarthritis or an osteoarthritis-like condition do not need to have an activated partial thromboplastin time (aPTT) greater than 5 seconds, which is above the upper limit of normal (ULN).

[0201] Individuals with osteoarthritis or an osteoarthritis-like condition do not need to have alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) above 1.5xULN, or total bilirubin outside the normal range. Individuals with osteoarthritis or an osteoarthritis-like condition do not need to have a known allergy to or sensitivity to acetaminophen.

[0202] Individuals with osteoarthritis or an osteoarthritis-like condition do not need to have a clinically significant acute or chronic medical condition that would make the use of IA injections impossible or compromise human safety. Individuals with osteoarthritis or an osteoarthritis-like condition do not need to have a bleeding disorder.

[0203] Provided that the dose has been stable for more than three months prior to treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention, a person suffering from osteoarthritis or an osteoarthritis-like condition may be administered aspirin for cardioprotection at a maximum dose of 81 milligrams (mg) per day. Provided that the dose / regimen has been stable for six months prior to treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention, a person suffering from osteoarthritis or an osteoarthritis-like condition may be administered pharmacotherapy for depression, including SSRIs, SNRIs, NSRIs, or tricyclic antidepressants. A person suffering from osteoarthritis or an osteoarthritis-like condition may be administered treatment for side effects associated with treatment with the pharmaceutical composition of the present invention or treatment by the method of the present invention, or an emergency drug.

[0204] Persons with osteoarthritis or an osteoarthritis-like condition may not be receiving any of the following: oral NSAIDs, topical NSAIDs, capsaicin, lidocaine patches, topical therapies applied to the indicator knee, cannabinoids, aspirin in doses exceeding 325 mg per day, centrally acting analgesics, opioids, muscle relaxants, any IA injections in the indicator knee, cryo- or radiofrequency nerve ablation of the indicator knee, any investigational drug, device or biopharmaceutical, any immunomodulator, immunosuppressant, or chemotherapeutic agent or combination thereof. Persons with osteoarthritis or an osteoarthritis-like condition may not be receiving pregabalin or gabapentin. Persons with osteoarthritis or an osteoarthritis-like condition may not be receiving oxycodone, hydrocodone, codeine, morphine, or tramadol. Individuals with osteoarthritis or an osteoarthritis-like condition do not need to be administered cyclobenzaprine, tetrazepam, or diazepam. Individuals with osteoarthritis or an osteoarthritis-like condition do not need to be administered local anesthetics, corticosteroids, hyaluronic acid, platelet-rich plasma, stem cells, prolotherapy, or amniotic fluid injection.

[0205] Manufacturing method The present invention comprises: (a) culturing and continuously expanding host cells; (b) infecting the continuously expanded host cells from (a) with the helper-dependent adenovirus (HDAd) and helper virus of the present invention; (c) culturing the infected cells from (b); (d) harvesting and lysing the infected cells from (c) to produce cell lysates; (e) digesting the host cell DNA in the cell lysates from (d); (f) clarifying the cell lysates from (e); (g) ultracentrifugation of the clarified cell lysates from (f); (h) collecting the virus from the ultracentrifuged cell lysates from (g); (i) gradient ultracentrifugation of the virus sample from (h); and (j) (i The present invention provides a method for producing a pharmaceutical composition, comprising: collecting a virus from a virus sample obtained by gradient ultracentrifugation of (k)(j); performing ultracentrifugation of the virus sample at the same density of (l)(k); collecting a virus from a virus sample obtained by ultracentrifugation of (m)(l); collecting a virus from a virus sample obtained by ultracentrifugation of (n)(m); dialysis of the collected virus at the same density of (o)(n); collecting and diluting the dialyzed virus at (p)(o); formulating the diluted virus at (q)(p); and filtering the formulated virus at (r)(o).

[0206] The host cells used in the method for producing the pharmaceutical composition of the present invention may be a 116 cell line derived from HEK293 cells. The host cells can be expanded using CellStacks® (CS). The host cells can be continuously expanded in growth medium (DMEM supplemented with FBS, L-glutamine, and hygromycin B). After culturing and expanding 116 cells at 37°C and 5% CO2 to prepare one batch and one further 10-layer CellStack (CS10), infection can be initiated.

[0207] The helper virus used in the method for producing the pharmaceutical composition of the present invention may be the AdNG178 virus. The step of infecting the continuously expanded host cells of (a) with the helper-dependent adenovirus (HDAd) and helper virus of the present invention can be carried out at 37°C and 5% CO2. The step of infecting the continuously expanded host cells of (a) with the helper-dependent adenovirus (HDAd) and helper virus of the present invention can be carried out in a volume of 600 ml per CS10. The culture of infected cells can be carried out at 37°C and 5% CO2 for 24 hours. The culture of infected cells can be carried out in DMEM supplemented with FBS and L-glutamine.

[0208] The harvesting of infected cells may include (i) detaching and collecting infected cells containing used culture medium to obtain an untreated bulk harvest containing the helper-dependent adenovirus (HDAd) of the present invention; (ii) clarifying the untreated bulk harvest by centrifugation; (iii) discarding the supernatant and resuspending the cell pellet in a lysis buffer; and (iv) freezing the harvested cells at -65°C. The lysis buffer used in the method for producing the pharmaceutical composition of the present invention may be 100 mM Tis, 10% glycerol at pH 8.0.

[0209] The untreated bulk harvest of the method for producing the pharmaceutical composition of the present invention is characterized by undetectable levels of mycoplasma, undetectable levels of foreign viruses, and microbial levels of less than 10 CFU / mL for TAMC and less than 10 CFU / mL for TYMC.

[0210] The step of lysing infected cells to produce cell lysates may include freezing and thawing the resuspended cell pellet in a lysis buffer for at least two cycles, each freeze-thaw-thaw cycle comprising first placing the suspended cell pellet in a freezing bath and then in a warm water (37°C) bath.

[0211] The step of digesting DNA in cell lysates may include treating the cell lysates with benzonase to digest any remaining host cell DNA. The step of digesting DNA in cell lysates may also include adding diluted benzonase in a buffer containing 10 mM Tris and 10 mM MgCl2 before the cell lysates for digestion.

[0212] The step of clarifying the cell lysate may include centrifugation of the cell lysate.

[0213] The step of ultracentrifugation of the cell lysate may include performing three rounds of cesium chloride (CsCl) ultracentrifugation to separate the helper-dependent adenovirus (HDAd) of the present invention from impurities based on specific gravity. The helper-dependent adenovirus (HDAd) collected from the rounds of ultracentrifugation of the cell lysate can be concentrated.

[0214] The concentrated helper-dependent adenovirus (HDAd) derived from the ultracentrifugation process of the method for producing the pharmaceutical composition of the present invention can be dialyzed to further remove impurities, including CsCl and potential residual hygromycin B. Dialysis can be performed in a formulation buffer containing 5% sucrose w / v, 0.5% ethanol v / v, 75 mM sodium chloride, 10 mM L-histidine, 10 mM Tris, 1.0 mM magnesium chloride, 0.02% polysorbate 80 v / v, and 100 μM EDTA. The dialysis process can be carried out four times. The dialyzed virus can be collected and diluted in the formulation buffer.

[0215] The formulation and dilution steps may include diluting the purified helper-dependent adenovirus (HDAd) to a desired target concentration in a formulation buffer. The formulation buffer contains 5% sucrose w / v, 0.5% ethanol v / v, 75 mM sodium chloride, 10 mM L-histidine, 10 mM Tris, 1.0 mM magnesium chloride, 0.02% polysorbate 80 v / v, and 100 μM EDTA. The formulation of the virus is sterile filtered through a 0.22 μm filter.

[0216] The “therapeutic effective dose” or “effective dose” of the adenovirus-based biological delivery and expression system of the present invention generally refers to the amount required to achieve a therapeutic objective. As stated above, this may be complete or partial recovery from osteoarthritis or a degenerative condition in the joints of a human subject requiring it. As stated above, this may be partial or complete prevention of the development of osteoarthritis or a degenerative condition in the progression of osteoarthritis in the joints of a human subject requiring it. The amount required for administration will further depend on the binding affinity of the fusion protein to its specific target, and also on the rate at which the administered fusion protein is depleted from the free volume of the other subject to which it is administered. The general range for therapeutic effective administration of the adenovirus-based biological delivery and expression system of the present invention is, but is not limited to, about 1.4 × 10⁻⁶. 8 ~1.4×10 12 Genome copy (GC) / ml is also acceptable.

[0217] Where used herein and in the appended claims, the singular “a,” “an,” and “the” refer to multiple subjects unless the context otherwise clearly indicates otherwise. “As needed” or “as required” means that the event or situation described thereafter may or may not occur, and that the description includes examples of the event or situation occurring and examples of the event or situation not occurring. For example, the phrase “a composition may include a combination as required” means that the composition may include a combination of different molecules, or it may not include a combination such that the description includes both the combination and the absence of the combination (i.e., the individual members of the combination). A range may be expressed herein as “about” one particular value and / or “about” another particular value. Where such a range is expressed, an alternative aspect includes one particular value and / or the other particular value. Similarly, where a value is expressed as an approximation, the use of the antecedent “about” will be understood to mean that a particular value forms an alternative aspect. It will be further understood that each endpoint of the range is significant in relation to the other endpoints, and independent of the other endpoints.

[0218] As used herein, the term “cell line” refers to a population of cells capable of growing and dividing continuously or over a long period in vitro. It is further known in the art that spontaneous or induced changes in karyotype may occur during the preservation or introduction of such clonal populations. Thus, cells derived from a cell line referred to may not be exactly identical to the ancestral cells or culture, and the cell line is referred to as containing such variants. In some embodiments, the terms “HEK293 cells,” “293 cells,” or their grammatical equivalents are used interchangeably herein and refer to the host / packing cell line used in the methods disclosed herein. This disclosure relates, for example, to the following: [1] A pharmaceutical composition comprising an adenovirus-based biological delivery and expression system for the treatment of osteoarthritis or osteoarthritis conditions in human joints, or for the prevention of such conditions in humans identified as being at risk of developing osteoarthritis or osteoarthritis conditions, The adenovirus-based biological delivery and expression system includes a helper-dependent adenovirus vector genome copy (GC) containing the nucleic acid sequence encoding the human interleukin-1 receptor antagonist (IL-1Ra) protein, left and right reverse terminal repeats, adenovirus packaging signals, and non-viral and non-coding stuffer nucleic acid sequences. The expression of the human IL-1Ra gene is regulated by an NF-κB-inducible promoter located upstream of the reading frame of the nucleic acid sequence encoding the human IL-1Ra protein. The nucleic acid sequence of the adenovirus-based biological delivery and expression system, including the promoter, the nucleic acid sequence encoding IL-1Ra, the left and right reverse terminal repeats, the adenovirus packaging signal, and the non-viral non-coding stuffer nucleic acid sequence, is at least 95% homologous to the nucleic acid sequence of Sequence ID No. 7. Adenovirus-based biological delivery and expression systems produce 1.4 × 10⁶ units per milliliter. 8 ~1.4×10 12 A pharmaceutical composition containing a GC (GC / ml) helper-dependent adenovirus vector. [2] The pharmaceutical composition according to [1], wherein the nucleic acid sequence of an adenovirus-based biological delivery and expression system, comprising a promoter, a nucleic acid sequence encoding IL-1Ra, left and right reverse terminal repeats, an adenovirus packaging signal, and a non-viral non-coding stuffer nucleic acid sequence, is at least 99% homologous to the nucleic acid sequence of Sequence ID No. 7. [3] The pharmaceutical composition according to [1], wherein the nucleic acid sequence of an adenovirus-based biological delivery and expression system comprises a promoter, a nucleic acid sequence encoding IL-1Ra, left and right reverse terminal repeats, an adenovirus packaging signal, and a non-viral non-coding stuffer nucleic acid sequence, the nucleic acid sequence comprising the nucleic acid sequence of Sequence ID No. 7. [4] The pharmaceutical composition according to [1], wherein the IL-1Ra in the nucleic acid sequence of an adenovirus-based biological delivery and expression system comprises the nucleic acid of SEQ ID NO: 4. [5] The pharmaceutical composition according to [4], wherein the nucleic acid described in SEQ ID NO: 4 expresses a human IL-1Ra protein having an amino acid sequence that is at least 95% homologous to SEQ ID NO: 6. [6] Adenovirus-based biological delivery and expression systems a)1.4×10 9 ~1.4×10 12 ; b)1.4×10 9 ~1.4×10 11 ;or c)1.4×10 9 ~1.4×10 10 GC / ml The pharmaceutical composition according to [1] above, comprising: [7] Adenovirus-based biological delivery and expression systems are available for 1.4 × 10⁶ 9 ~5.6×10 9 The pharmaceutical composition according to [6], comprising GC / ml. [8] Adenovirus-based biological delivery and expression systems are available for 1.4 × 10⁶ 10 ~5.6×10 10 The pharmaceutical composition according to [6], comprising GC / ml. [9] Adenovirus-based biological delivery and expression systems are available for 1.4 × 10⁶ 11 ~5.6×10 11 The pharmaceutical composition according to [6], comprising GC / ml.

[10] A pharmaceutical composition according to any one of the claims [6] to [9], comprising an adenovirus-based biological delivery and expression system, comprising a dose volume of up to 5 ml.

[11] Adenovirus-based biological delivery and expression systems are available for 7 × 10⁶ 9 ~2.8×10 10 The pharmaceutical composition according to [7], comprising the total dose of GC.

[12] Adenovirus-based biological delivery and expression systems are available for 7 × 10⁶ 10 ~2.8×10 11 The pharmaceutical composition according to [8], comprising the total dose of GC.

[13] Adenovirus-based biological delivery and expression systems are available for 7 × 10⁶ 11 ~2.8×10 12 The pharmaceutical composition according to [9], comprising the total dose of GC.

[14] The pharmaceutical composition described in [1] above, which is formulated for intratendinous, intramuscular, intraarticular, or subacromial injection into a human joint.

[15] A pharmaceutical composition as described in [1] above, formulated for intra-articular injection into a human joint.

[16] A method for infecting articular cells from one or more osteoarthritis-affected joints of a human suffering from osteoarthritis or an osteoarthritis-affected condition with an adenovirus-based biological delivery and expression system, a) a step of infecting articular cells of a human joint affected by osteoarthritis that requires it with a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system as described in any one of the preceding paragraphs [1] to

[15] ; b) A step of expressing IL-1Ra in a target region within a joint affected by osteoarthritis. Methods that include...

[17] The method according to

[16] , wherein arthrocytes are once infected with an adenovirus-based biological delivery and expression system.

[18] The method according to

[16] , wherein arthrocytes are infected with an adenovirus-based biological delivery and expression system two or more times.

[19] The method according to

[18] , wherein articular cells are infected with an adenovirus-based biological delivery and expression system two or more times, and each infection comprises a helper-dependent adenovirus vector with a different number of genomic copies.

[20] The method according to

[18] , wherein arthrocytes are infected with an adenovirus-based biological delivery and expression system two or more times, and each infection comprises a helper-dependent adenovirus vector of the same number of genomic copies.

[21] The method according to any one of the claims

[18] to

[20] , wherein arthrocytes are infected with an adenovirus-based biological delivery and expression system two or more times, and each infection is carried out in the same osteoarthritis-affected joint of a human.

[22] The method according to any one of the claims

[18] to

[20] , wherein, when articular cells are infected with an adenovirus-based biological delivery and expression system two or more times, all second and subsequent infections are carried out in an osteoarthritis-affected joint of a different human than the osteoarthritis-affected joint in which the previous infections occurred.

[23] The method according to any one of the claims

[16] to

[22] , wherein the infection of arthrocytes includes intra-articular, intratendinous, intramuscular, or subacromial injection.

[24] c) A step of monitoring the treatment or progression of osteoarthritis or the osteoarthritis state in the osteoarthritis-affected joint after the expression of IL-1Ra in (b). The method described in

[16] , further comprising:

[25] (d) If monitoring in (c) indicates that osteoarthritis or an osteoarthritis condition in a human joint is not under control or treatment, the step of continuing to administer the adenovirus-based biological delivery and expression system in that amount to the osteoarthritis-affected joint in (a); or If monitoring in (e)(c) indicates progression of osteoarthritis or an osteoarthritis-like condition in a human joint, the process involves further adjusting the genome copy number of the helper-dependent adenovirus vector in the adenovirus-based biological delivery and expression system and administering it to the osteoarthritis-affected joint in (a). The method described in

[24] , further comprising:

[26] An adenovirus-based biological delivery and expression system for the treatment of osteoarthritis or osteoarthritis conditions in human joints, or for the prevention of such conditions in humans identified as being at risk of developing osteoarthritis or osteoarthritis conditions, The adenovirus-based biological delivery and expression system includes a helper-dependent adenovirus vector genome copy (GC) containing a nucleic acid sequence encoding a human interleukin-1 receptor antagonist (IL-1Ra), left and right reverse terminal repeats, an adenovirus packaging signal, and non-viral and non-coding stuffer nucleic acid sequences. The expression of the human IL-1Ra gene is regulated by an NF-κB-inducible promoter located upstream of the reading frame of the nucleic acid sequence encoding human IL-1Ra. The nucleic acid sequence of the adenovirus-based biological delivery and expression system, including the promoter, the nucleic acid sequence encoding IL-1Ra, the left and right reverse terminal repeats, the adenovirus packaging signal, and the non-viral non-coding stuffer nucleic acid sequence, is at least 95% homologous to the nucleic acid sequence of Sequence ID No. 7. Adenovirus-based biological delivery and expression systems were isolated from host cells infected with helper-dependent adenovirus vectors and helper viruses. Adenovirus-based biological delivery and expression systems a) 1.4 × 10⁻¹⁶ per milliliter of synovial fluid in human joints 8 ~1.4×10 12 GC (GC / ml) helper-dependent adenovirus vector; b) Helper virus particles less than 15%; c) Less than 10% empty capsids; d) Host cell proteins at a concentration of 100 μg / ml or less; e) Host cell nucleic acids less than 20 ng / ml; f) Endotoxins less than 35 EU / ml; and g)300GC / TCID 50 The following ratios of virus particles to infection units Adenovirus-based biological delivery and expression systems, including those mentioned above.

[0219] The present disclosure is further illustrated by the following embodiments, which should not be construed as limiting. [Examples]

[0220] [Example 1] Comparison of transduction efficiency between helper-dependent adenovirus vectors and adeno-associated virus vectors in mouse joints. Objective of the study: This specification describes a study conducted to evaluate and compare the transduction efficiency of HDAd vectors and AAV vectors in mouse joints.

[0221] Methods: The test items in this study were HDAd (Ad5 serotype) and AAV vectors pseudotyped with AAV2, AAV2.5, and AAV6 capsids encoding GFP under the control of a cytomegalovirus (CMV) promoter, all to mark transduced cells. Eight-week-old male FVB / N mice were divided into five groups, each containing two mice. The vector was administered intra-articularly (IA) to both knee joints in a dose volume of 5 μL. Group 1 received 5 × 10⁶ mice. 9 VP / Knee HDAd-GFP was administered. Groups 2-4 received 5 × 10 9 The mice received AAV2-GFP, AAV2.5-GFP, and AAV6-GFP in vector genome (vg) / knee doses, respectively. Group 5 received phosphate-buffered saline (PBS) as a medium. After one week, the mice were euthanized, and the knee joints were prepared (decalcified and paraffin-embedded) for histological analysis, sectioned, and stained with fluorescently labeled anti-GFP antibody.

[0222] Results: HDAd-GFP injection resulted in robust GFP staining, which was somewhat variable among individual injected joints within the group. The staining appeared to be primarily localized in synovial tissue (Figure 3A). GFP expression was weaker in all AAV-injected knees compared to joints injected with HDAd. Among the AAV groups, AAV6 appeared to produce the strongest GFP expression. No staining was observed in joints injected with the medium (Group 5), although this is not shown.

[0223] Conclusion: HDAd(Ad5) robustly transduced arthrocytes after IA injection. In particular, synovial surface cells appeared to be transduced. In comparison, transduction with serotypes 2, 2.5, and 6 AAV vectors appeared to be less efficient.

[0224] [Example 2] HDAd mediates the long-term expression of marker genes in the joints. Objective of the study: This specification describes a study conducted to determine long-term gene expression in the joints of mice injected intra-articularly with the helper-dependent adenovirus vector (HDAd) of the present invention and, for comparison, with a first-generation adenovirus (Ad) vector expressing firefly luciferase (luc) under the control of the CMV promoter, for up to one year.

[0225] Method: Put 10 8 Viral particles (VPs) were injected intra-articularly into the knee joints of four mice per group, using either a luciferase-expression helper-dependent (HDAd-luc) or corresponding first-generation (Ad-luc) adenovirus vector. Three days later, the mice were imaged using the MS200 series imaging system (Caliper Life Sciences, Hopkinton, MA). After luciferase expression in the mice, repeated bioluminescence imaging was performed and quantified using Living Image 2.5 software (Caliper Life Sciences).

[0226] Results: Potent bioluminescent signals were detected in joints injected with both HDAd-luc and Ad-luc adenovirus vectors. A potent initial luc signal was detected 3 days after injection of both vectors (Figure 4A). Expression subsequently decreased for both vectors, becoming undetectable after 1 month for the first-generation vector Ad-luc (Figure 4B). However, HDAd-luc luciferase expression stabilized at day 10 and maintained this level for 380 days.

[0227] Conclusion: Helper-dependent and first-generation adenovirus vectors mediate similar levels of marker gene expression. Helper-dependent adenovirus vectors mediate long-term marker gene expression in joints.

[0228] [Example 3] HDAd transduces synovial cells after intra-articular injection. 10 8 or 109 This specification describes a study conducted to evaluate in detail the HDAd transduction in mouse joints after intra-articular injection of VP LacZ-expressing HDAd. Positive LacZ expression was observed in 10 9 This was observed in both synovial and chondrocytes of joints infected with VP LacZ-expressing HDAd (Figure 5B), 10 8 No staining was observed in chondrocytes in joints infected with VP (Figure 5A). The livers of these animals were analyzed to assess whether the virus escaped from the joints or spilled during injection. Most importantly, detectable vector concentrations above background levels could not be measured by quantitative PCR (data not shown). Therefore, the vector specifically localizes and remains in the joints, which suggests minimal side effects and is highly beneficial in the treatment or prevention of osteoarthritis.

[0229] [Example 4] Cells infected with HDAd-Il-1Ra secrete IL-1Ra. (Injection) Objective of the study: This specification describes a study conducted to generate HDAd expressing IL-1Ra under the control of an inflammation-sensitive NF-KB5-ELAM promoter and to test its function in vitro.

[0230] Methods: Human embryonic kidney cells (HEK293) were infected with 100 VP / cell of HDAd-IL-1Ra, HDAd-GFP, or mock. Two days later, IL-1Ra ELISA was performed using the cell culture supernatant. A concentration of approximately 700 pg / ml was measured in cells infected with HDAd-IL-1Ra, but IL-1Ra was undetectable in the supernatant of HDAd-GFP or mock-infected cells. To induce an inflammatory response, lipopolysaccharide (LPS, 100 μg / ml) was added to half of the sample, and the IL-1Ra concentration was re-determined one day later (day 4). The level in the HDAd-IL-1Ra sample increased to approximately 1600 pg / ml, but cells that were not induced produced less IL-1Ra compared to the previous day. IL-1Ra expression was not detected in any of the control samples (HDAd-GFP and mock).

[0231] Results: High levels of IL-1Ra were measured in the supernatant of HDAd-Il-1Ra-infected cells on day 3 (Figure 6). Induction of inflammation by lipopolysaccharide (LPS) resulted in a dramatic increase in IL-1Ra concentration compared to samples that were not induced. IL-1Ra was not detected in uninfected samples (mock) or samples infected with the control vector (HDAd-GFP). High levels of IL-1Ra were measured in the supernatant of synovial cells infected with the helper-dependent adenovirus vector of the present invention (HDAd). As shown in Figure 6, induction of inflammation by lipopolysaccharide (LPS) resulted in a dramatic increase in IL-1Ra concentration compared to samples that were not induced. IL-1Ra was not detected in uninfected samples (mock) or samples infected with the control vector (HDAd-GFP).

[0232] Conclusion: The results disclosed herein demonstrate that cells infected with HDAd-mIl-1Ra can produce high levels of Il-1Ra. It further indicates that IL-1Ra is efficiently secreted from these cells, and that an inflammatory state activates the NF-KB5-ELAM promoter, leading to increased IL-1Ra levels.

[0233] Acute injury to the anterior cruciate ligament (ACL) is a common cause of post-traumatic osteoarthritis (OA) in humans, and ACL transection (ACLT) in rats and mice is an established animal model of trauma-induced OA. FX201, an intra-articular (IA) gene therapy candidate based on a helper-dependent adenovirus (HDAd) designed to induce the production of interleukin (IL)-1 receptor antagonist (IL-1Ra) in the presence of inflammation, is under development as a potential therapeutic agent for OA. Described herein are studies demonstrating the efficacy of treatment with FX201 or its equivalent HDAd-mIL-1Ra in the prevention and treatment of OA.

[0234] [Example 5] HDAd-Il-1Ra prevents the development of OA in mice. Objective of the study: This specification describes a study conducted to evaluate whether an HDAd vector expressing mouse IL-1Ra under the control of an NF-κB-inducible promoter upregulated by inflammatory stimuli (HDAd-mIL-1Ra) can prevent the progression of OA in a mouse model of the disease. HDAd-mIL-1Ra has the same vector scaffold as FX201 but encodes a mouse-specific IL-1Ra transgene (mIL-1Ra).

[0235] Methods: To evaluate whether HDAd expressing IL-1Ra can prevent the development of osteoarthritis (OA), HDAd-IL-1Ra or a GFP-expressing control vector (HDAd-GFP) was intra-articularly injected into the knee joints of mice. 8VP's HDAd-IL-1Ra, HDAd-GFP, or mock ligament was injected intra-articularly into the knee joint. Two days after injection, cruciate ligament dissection was performed to induce OA development. This osteoarthritis model was developed by Dr. Brendan Lee's research group and validated in several experiments (Ruan, Z., Dawson, B., Jiang MM, Gannon, F., Heggeness, M., Lee, B. (2012). Quantitative volumetric imaging of murine osteoarthritic condition cartilage by phase contrast micro-computed tomography, submitted). This model involves anterior and posterior cruciate ligament dissection of the knee joint and leads to the development of severe OA. Mice were sacrificed one month after OA induction, and the joints were histologically prepared and stained with safranin O. The development of osteoarthritis (OA) was scored by blinded pathologists according to OARSI (International Association for Osteoarthritis) criteria (assigned scores on a scale of 1 to 6, where 1: no signs of OA, and 6: maximum OA).

[0236] Results: Joints treated with HDAd-IL-1Ra had significantly lower OA scores than joints treated with HDAd-GFP or left untreated, suggesting that HDAd-IL-1Ra prevented the development of OA (Figure 7). Since the mean OA score was comparable to that of the untreated group, the control vector HDAd-GFP was considered to have no effect on the development of OA.

[0237] Conclusion: The results disclosed herein indicate that infecting mouse joints with HDAd-IL-1Ra prevents the development of osteoarthritis (OA).

[0238] [Example 6] Prevention of osteoarthritis in mice using locally administered HDAd-mIL-1Ra. Objective of the study: This specification describes a study to evaluate whether an HDAd vector expressing mouse IL-1Ra under the control of an NF-κB-inducible promoter upregulated by inflammatory stimuli (HDAd-mIL-1Ra) can prevent the progression of OA in a mouse model of the disease. HDAd-mIL-1Ra has the same vector scaffold as FX201 but encodes a mouse-specific IL-1Ra transgene (mIL-1Ra).

[0239] Method: The test item HDAd-mIL-1Ra (Group 1) or the control vector HDAd-GFP (Group 2) was subjected to 10 doses as shown in Table 1. 8 8-week-old male FVB / N mice were injected with IA into both knees at the VP / knee dose (5 mice per group) and a dose volume of 3 μL. A control group (Group 3) received a medium (PBS). Two days later, osteoarthritis (OA) was induced by rupture of the cruciate ligaments above all injected knees. Thirty days later, the mice were euthanized, and histological evaluation of the treated joints was performed. To assess cartilage damage, sections from the lateral compartment were scored according to the OARSI histological grading system, with scores for the tibia and femur added together (the scoring system is detailed in Table 3). To assess synovitis, sections were scored using the 3-point scale described in Table 4; osteophytes were scored as present or absent in the tissue section and expressed as the percentage of joints with osteophytes for all joints analyzed.

[0240] [Table 1]

[0241] Results: The mean OARSI scores for both the media group (Group 3) and Group 2 treated with the control HDAd-GFP vector were approximately 8.5, and were not significantly different from each other (Figure 8A). However, the mean OA score for the group administered with HDAd-mIL-1Ra (Group 1) was approximately 6, which was significantly lower than the scores of the media and HDAd-GFP groups, indicating that there were significantly fewer histological OA characteristics in the joints treated with HDAd-mIL-1Ra. No difference in synovitis scores was observed between the media and HDAd-GFP groups (Figure 8B). The mean synovitis score for Group 1 treated with HDAd-mIL-1Ra was lower than the mean scores of the media and HDAd-GFP groups, although this was not statistically significant. As shown in Figure 8C, fewer osteophytes were detected in the HDAd-GFP group compared to the media-treated group. Fewer osteophytes were observed in Group 1 treated with HDAd-mIL-1Ra compared to both Groups 2 and 3.

[0242] Conclusion: The results described herein indicate that media-treated mice with cruciate ligament rupture developed severe OA within one month, as indicated by high OARSI scores and the presence of synovitis and osteophytes. HDAd vectors expressing GFP did not significantly alter the histological features of OA. This indicates that the HDAd vector itself has no effect on disease progression. HDAd expressing mouse IL-1Ra showed improved OA pathology compared to media and HDAd-GFP-treated mice, as indicated by significantly improved OARSI scores, a tendency toward lower synovitis scores (not statistically significant), and fewer osteophytes in the joints, compared to HDAd-GFP and media-treated groups.

[0243] [Example 7] HDAd-mIL-1Ra treats osteoarthritis in mouse models of the disease. Objective of the study: This specification describes a study to evaluate the efficacy of HDAd-mIl-1Ra in the treatment of OA in the mouse disease model described above.

[0244] Methods: OA was induced in the mouse knee joint by cruciate ligament dissection, and the disease was allowed to develop. Two weeks after dissection, the mice were given 10 8 VP was injected intra-articularly with HDAd-IL-1Ra, HDAd-GFP, or a mock. Six weeks later, mice were sacrificed, histologically prepared, sectioned, and stained with safranin O. Blinded pathologists assessed the level of OA according to OARSI (International Osteoarthritis Society) criteria (assigning scores on a scale of 1-6, 1: no signs of OA, 6: maximum OA). The joints were further evaluated by microcomputed tomography (μCT) analysis. This technique combines high-resolution (up to 0.5 μm) X-ray CT scans with phase-contrast lenses to visualize cartilage in small animal joints. Three-dimensional reconstruction of the joints and computerized histological analysis tools can be used to quantify several cartilage parameters, such as volume and surface area. The entire knee joints of mice treated as described above were fixed in electron microscope fixative and embedded in paraffin. They were scanned using an X-ray micro-XCT scanner (Xradia, Pleasanton, CA, USA) and visualized at a resolution of 4 μm. Computerized 3D reconstruction of the joints was performed, and the volume and surface area of ​​the cartilage were semi-automatically quantified using TRI BON software (RATOC System Engineering, Tokyo, Japan).

[0245] Results: Using a model, we evaluated whether HDAd-IL-1Ra could efficiently treat OA. Therefore, OA was induced by cruciate ligament dissection (except for the undissected group), and OA was allowed to develop over two weeks. Then, HDAd-IL-1Ra, a control vector (HDAd-GFP), or a medium was injected, and after another six weeks, the mice were sacrificed and their joints analyzed. Both HDAd-GFP-treated and uninjected mice developed OA to a similar level, with a mean score of approximately 4.5 (Figure 9A). However, HDAd-IL-1Ra-treated mice had significantly lower OA scores compared to the HDAd-GFP and mock-treated groups. No significant difference was observed between HDAd-IL-1Ra-treated mice and undissected (no OA) mice, suggesting effective treatment or prevention of the disease. HDAd-IL-1Ra-treated joints showed significantly higher cartilage volume compared to HDAd-GFP and mock-treated joints (Figure 9B). No significant difference was observed between the HDAd-IL-1Ra group and the transfected (no OA) group. Furthermore, cartilage surface area was significantly larger in HDAd-IL-1Ra-treated mice compared to the HDAd-GFP and mock groups (Figure 9C), but no significant difference was observed between HDAd-IL-1Ra-treated joints and transfected (non-OA) joints.

[0246] Conclusion: The results disclosed herein demonstrate that infection with HDAd-IL-1Ra effectively treats OA in mice. The results disclosed herein also demonstrate that infection with HDAd-IL-1Ra prevents the loss of cartilage surface area and volume caused by the induction of OA compared to untreated control mice.

[0247] [Example 8] Locally administered HDAd-mIL-1Ra for the treatment of osteoarthritis in skeletal mature mice Objective of the study: This specification describes a study to evaluate whether HDAd-mIL-1Ra can slow the progression of OA in skeletal mature mice. HDAd-mIL-1Ra has the same vector skeleton as FX201 but encodes a mouse-specific IL-1Ra transgene.

[0248] Methods: OA was induced in 60 12-week-old male FVB / N mice by rupture of the cruciate ligament in both knee joints (Day 0). 72 hours later (Day 3), the test item HDAd-mIL-1Ra (Group 1) or the control vector HDAd-GFP (Group 2) was administered to 10 9 VP was administered via IA injection into both knees at doses and dose volumes of 5 μL (16 mice per group) (Table 2). Group 3 received IA injection of PBS (8 mice per group). Group 4 included 12 control mice that underwent sham surgery without any treatment. Hot plate nociception analysis was performed on all mice from day 57 to 59 to assess thermal hyperalgesia. The latency period to hindlimb flipping and hindlimb licking or jumping was recorded as minor and major response times, respectively. Mice were sacrificed on day 60. The left and right knees of each animal were processed for histological analysis and phase-contrast microCT analysis, respectively.

[0249] To assess cartilage damage, sections from the lateral compartment were scored in a blinded manner according to the OARSI tissue grading system, with scores for the tibia and femur added together (the scoring system is detailed in Table 3). To assess synovitis, sections were scored using the 3-point scale described in Table 4; osteophytes were scored as present or absent in the tissue section and expressed as the percentage of joints with osteophytes for all joints analyzed. Using micro-CT imaging, cartilage volume and the area of ​​bone covered by cartilage were analyzed by blinded evaluators.

[0250] [Table 2]

[0251] [Table 3]

[0252] [Table 4]

[0253] Results: Compared to the groups injected with HDAd-GFP (8.21; Group 2) or the media (7.13; Group 3), animals treated with HDAd-mIL-1Ra showed a tendency toward lower mean tissue scores for cartilage damage (5.75; Group 1); however, the difference was not statistically significant. All treatment groups had significantly higher scores compared to the sham surgery group (1.25; Group 4). In hot plate nociception analysis, HDAd-mIL-1Ra treatment resulted in a significantly longer mean minor response time (4.14 seconds) compared to HDAd-GFP (2.93 seconds) and media treatment (2.64 seconds). The minor response time in HDAd-mIL-1Ra-treated mice was not significantly different from that of healthy mice in the sham group, which indicates protection from thermal hyperalgesia. Compared to the HDAd-GFP group (8.64 seconds) and the media group (8.97 seconds), HDAd-mIL-1Ra-treated mice showed a tendency toward longer major response times in hot plate nociceptive analysis (12.65 seconds), but the difference was not statistically significant.

[0254] Synovitis scores were similar in joints treated with HDAd-mIL-1Ra, HDAd-GFP, or the media. Similarly, the effect of the HDAd-mIL-1Ra vector on the number of osteophytes was not clear. Synovitis scores and the number of osteophytes were significantly lower in the sham control group compared to the group in which OA was induced.

[0255] Micro-CT imaging showed significantly lower cartilage volume and cartilage-covered bone area in the medium and HDAd-GFP groups compared to healthy joints in Group 4 (Figure 10). Cartilage volume and surface area in the HDAd-mIL-1Ra group were significantly higher than in the medium and HDAd-GFP groups and did not differ significantly from healthy joints in Group 4.

[0256] In hot plate nociception analysis, HDAd-mIL-1Ra treatment resulted in a significantly longer mean minor response time (4.14 seconds) compared to HDAd-GFP (2.93 seconds) and media treatment (2.64 seconds). The minor response time in HDAd-mIL-1Ra-treated mice was not significantly different from that of healthy mice in the placebo group, indicating protection from thermal hyperalgesia. Compared to the HDAd-GFP group (8.64 seconds) and media group (8.97 seconds), a tendency toward a longer major response time was observed in HDAd-mIL-1Ra-treated mice in hot plate nociception analysis (12.65 seconds), but the difference was not statistically significant.

[0257] Conclusion: The results disclosed herein indicate that 12-week-old skeletal mature mice that underwent cruciate ligament rupture and were injected with the medium developed severe OA over a 60-day course, as evidenced by significantly higher OARSI and synovitis scores, a greater number of osteophytes, significantly lower cartilage volume and cartilage-covered bone area, and significantly shorter times to minor and major responses in the hot plate nociception assay, compared to healthy sham control mice. A control HDAd vector expressing GFP had no effect on any of these parameters.

[0258] A tendency toward lower OARSI scores was observed in mice treated with HDAd-mIL-1Ra compared to animals treated with HDAd-GFP or the media, but the effect of the treatment on synovitis score or the number of osteophytes was not clear in this OA model. Micro-CT analysis of HDAd-mIL-1Ra-treated joints showed significantly higher cartilage volume and cartilage-covered bone area compared to joints treated with the control vector HDAd-GFP or the media. Mice treated with HDAd-mIL-1Ra showed a tendency toward a reduced thermal nociceptive response compared to controls, but the difference was not statistically significant. These data suggest that in this severe model of OA in skeletal mature mice, IA injection of HDAd-mIL-1Ra slows cartilage degeneration and improves pain parameters. The fact that efficacy endpoints assessed by micro-CT were statistically significant, while histological assessments did not show statistically significant differences, may be due to the increased sensitivity of the former analytical method.

[0259] [Example 9] The effect of HDAd-rat IL-1Ra in a rat model of posterior cruciate ligament dissection (ACLT) in osteoarthritis. Study Objective: A study is described herein to evaluate the efficacy of HDAd-rat IL-1Ra as a surrogate for FX201 when administered to rats as a single intra-articular injection one week after ACLT surgery. The objective of this study was to evaluate the efficacy of HDAd-rat IL-1Ra (a rat surrogate for FX201, an adenovirus-based biological delivery and expression system incorporated herein by reference, U.S. Patent No. 10,301,647), a helper-dependent adenovirus encoding a rat interleukin-1 receptor antagonist protein, when administered to rats by a single intra-articular injection one week after ACLT surgery.

[0260] Methods: Table 5 summarizes the experimental procedures applicable to pathological investigations.

[0261] [Table 5]

[0262] In Table 5 shown above, IA is an intra-articular injection; TI is a test item. The surgery date was assumed to be day 7.

[0263] At the conclusion of the study at 12 weeks post-surgery, the right knee joint was harvested from all rats, pinned to a paraffin block, maintained at an angle of approximately 110°, and fixed in 10% neutral buffered formalin (NBF). These knees were used for micro-CT imaging and histopathological evaluation.

[0264] For histopathological evaluation, the entire right knee joint was decalcified, embedded in paraffin wax, and coronally sectioned. Three sections were stained with safranin-O fast green (SOFG), and one section was stained with H&E. The cartilage / bone and synovial membranes were individually scored using a semi-quantitative grading system (OARSI), as detailed in Attachment 1. Individual scores from representative sections were recorded in an Excel spreadsheet, and the sum of all individual scores generated an overall composite score.

[0265] A total of 46 Sprague Dawley rats were assigned to one of four test groups: high-dose HDAd-rat IL-1Ra (ACLT / HDAd-rat IL-1Ra; 2.4 × 10⁻¹⁴). 8 Virus particles [VP] / dose; n=12), low dose HDAd-rat IL-1Ra (ACLT / HDAd-rat IL-1Ra; 3×10 7VP / dose (n=12), medium (ACLT / medium; n=12), or sham / untreated (n=10). Rats underwent ACLT surgery (except for sham animals) under isoflurane anesthesia on day 7. Seven days after surgery (day 1), rats received a single IA injection of HDAd-rat IL-1Ra or medium into the right knee joint under anesthesia. At week 12, animals were sacrificed, the entire right knee joint was harvested, and analyzed for histopathology. For histopathological evaluation, the entire right knee joint was stained with safranin O fast green (SOFG) as well as hematoxylin and eosin, and evaluated using a semi-quantitative grading system (OARSI score) to score cartilage / bone and synovial membrane, respectively. Individual scores were recorded, and composite scores for each parameter were generated by summing all individual scores.

[0266] Results: Anterior cruciate ligament dissection (ACLT) successfully induced microscopic changes in osteoarthritis (OA) in the knee joint of all rats 12 weeks after surgery. 3 × 10⁻¹⁰ saturates at 1 week post-ACLT in Sprague Dawley rats. 7 and 2.43 × 10 8 A single intra-articular injection of HDAd-rat IL-1Ra at VP / dose resulted in a dose-dependent reduction in the incidence / severity of OA-related lesions to cartilage / bone and synovium, with corresponding median and mean scores being lower in the ACLT-treated group compared to the ACLT-treated group.

[0267] In sham-operated rats, microscopic changes at week 12 were limited to a low incidence of surface articular cartilage changes graded as minimal in severity, as assessed by lesional fibrosis / tears / cracks, chondrocyte loss, and / or surface irregularities showing SOFG staining. All ACLT-operated rats developed microscopic changes in OA, ranging from minimal to severe in one or more examined articular compartments at week 12. These changes ranged from minimal to severe and consisted of surface irregularities to complete articular cartilage fibrosis / tears / cracks / loss, SOFG staining loss, clonalization, and / or chondrocyte loss.

[0268] In sham-operated rats, microscopic changes at week 12 were limited to a low incidence of surface articular cartilage changes graded as minimal in severity, as assessed by lesion fibrosis / tears / fissures, chondrocyte loss, and / or surface irregularities showing SOFG staining. All ACLT-operated rats developed microscopic changes in OA, ranging from minimal to severe in one or more examined articular compartments at week 12. Among ACLT-operated rats, HDAd-rat IL-1Ra resulted in a dose-dependent reduction in the composite score for cartilage / bone compared to the medium (Figure 11). These reductions are likely attributable to the reduced severity of structural changes and chondrocyte loss associated with HDAd-rat IL-1Ra treatment compared to the medium (Figure 12). Compared to the control group, HDAd-rat IL-1Ra treatment also showed a slight decrease in the severity of SOFG staining loss and the incidence of clonal formation (Figure 12). Compared to ACLT-operated rats in the control group, HDAd-rat IL-1Ra-treated rats showed a dose-related decrease in the median and mean composite scores of microscopic changes in synovial OA. All synovial microscopic findings were minimal in severity and included synovial cell proliferation / hypertrophy, lymphoplasmacytic infiltration rarely forming aggregates / folliculars, villous hyperplasia with fibroblast / vascular proliferation, and cartilage / bone debris (Figure 13). As shown in Figures 11 and 12, respectively, the dose-related decrease in median and mean composite scores in HDAd-rat IL-1Ra-treated rats at week 1 compared to ACLT-operated rats in the control group was mainly due to the lower severity of structural changes and chondrocyte loss. Compared to animals derived from the ACLT / medium group, treated rats showed a slight reduction in the severity of SOFG stain loss and the incidence of clonal formation.

[0269] Conclusion: The results described herein demonstrate that a single dose of HDAd-rat IL-1Ra, a rat substitute for FX201, resulted in a dose-dependent reduction in the incidence and severity of OA-related lesions to cartilage / bone and synovial membrane at 12 weeks post-surgery; these results support the further development of FX201 as a potential therapeutic agent for OA.

[0270] [Example 10] Efficacy, safety, and in vivo distribution of FX201, a helper-dependent adenovirus gene therapy for the treatment of osteoarthritis in a rat model of anterior cruciate ligament rupture. Study Objective: This specification describes a study to evaluate the efficacy, safety, and biodistribution of HDAd-rat IL-1Ra, a rat substitute for FX201, when administered as a single intra-articular (IA) injection in a rat model of anterior cruciate ligament dissection (ACLT) in osteoarthritis, as well as the biodistribution of FX201.

[0271] Methods: Sprague-Dawley rats underwent ACLT surgery on the right knee (excluding dummy animals). To evaluate safety and vector biodistribution, rats received a single IA injection of HDAd-rat IL-1Ra, FX201, or the vector on the right knee 28 days post-surgery. Safety was evaluated in six test groups: HDAd-rat IL-1Ra (3.2 × 10⁸, 3.1 × 10⁸) 9 , or 4.3 × 10 10 The vector biodistribution was evaluated throughout the study in a cohort of male rats (N=144) equally assigned to GC / dose, ACLT / vector, ACLT / untreated, or sham / untreated. The biodistribution of the vector was assessed at days 8, 29, and 92 for HDAd-rat IL-1Ra (4.3 × 10⁶). 10 GC / dose) or FX201 (4.1 x 10 10 The study evaluated rats that underwent ACLT surgery and received GC / dose (n=12 per group at each time point [1:1 sex ratio]).

[0272] Results: In the efficacy studies described herein, HDAd-rat IL-1Ra showed a reduction in OARSI composite score compared to the medium. In safety studies, HDAd-rat IL-1Ra did not cause any adverse effects on mortality, body weight and food consumption, or clinicopathological or anatomical pathology at any dose tested. Anti-Ad5 titer increased with HDAd-rat IL-1Ra dose and decreased from day 29 to day 92. Similarly, dose-dependent anti-Ad5 T cell response decreased from day 29 to day 92 when assessed by interferon-γELISpot using splenocytes derived from HDAd-rat IL-1Ra-treated rats. In in vivo distribution studies, HDAd-rat IL-1Ra and FX201 were detectable up to day 92, with the highest concentration at the injection site. Both were detected sporadically at low levels in the liver, spleen, lungs, and bone marrow on days 8 and 29, but were not detected in the plasma or organs such as the brain, heart, and kidneys at any of the time points examined, confirming the absence of systemic circulation.

[0273] Conclusion: The results described herein indicate that, following a single local IA injection in rats, HDAd-rat IL-1Ra improved OA-related lesions to cartilage at 12 weeks post-surgery at all doses tested in the efficacy study. HDAd-rat IL-1Ra was well tolerated, and the no-adverse-action level (NOAEL) was 4.3 × 10⁶, the highest dose tested. 10 It is thought to be GC, and in the rat ACLT model of OA, the minimum effective dose (3.6 × 10) 7 The difference between GC / dose and NOAEL was 1000-fold. Furthermore, HDAd-rat IL-1Ra and FX201 did not enter the systemic circulation. These results described herein support further development of FX201; a Phase I trial in patients with knee OA is currently underway (NCT04119687).

[0274] [Example 11] In vitro expression of IL-1Ra from GQ-201 (HDAd-eqIL-1Ra) Objective of the study: This study describes a test to evaluate the level of IL-1Ra secreted by HEK293 (human embryonic kidney) cells infected with HDAd-eqIL-1Ra (referred to as GQ-201 in the study report) and to demonstrate that the NF-κB-inducible promoter encoded by the vector is functional, i.e., that inflammatory stimuli increase IL-1Ra expression. HDAd-eqIL-1Ra is an HDAd vector that expresses equine IL-1Ra under the control of the NF-κB promoter. It is equivalent to FX201 except that HDAd-eqIL-1Ra carries the equine version of IL-1Ra, as opposed to the human version present in FX201. Used as a control, the HDAd-GFP vector encodes green fluorescent protein (GFP) instead of IL-1Ra but shares the same scaffold as HDAd-eqIL-1Ra and FX201.

[0275] Methods: HEK293 cells were seeded in three replicates on a 12-well plate (250,000 cells per well) and incubated overnight in 1 mL of Minimum Essential Medium (MEM) (10%MEM) containing 10% fetal bovine serum. On the following day (day 0), the medium was replaced with 200 μL of the corresponding infection medium containing the viruses listed in Table 6. After incubating the cells for 1 hour, the infection medium or medium was aspirated and replaced with 10%MEM. On day 1, supernatant samples were taken from the wells and frozen at -20°C. On day 2, the medium in all wells was replaced with fresh 10%MEM. On day 3, supernatant samples were taken from the wells and frozen at -20°C. The medium from all wells was replaced with either 10%MEM containing 100 μg / mL of LPS or plain 10%MEM, as shown in Table 6. On day 4, the supernatant was collected and frozen at -20°C. The samples were analyzed using an enzyme-linked immunosorbent assay (ELISA) specific to equine IL-1Ra.

[0276] [Table 6]

[0277] Results: In groups 1A and 1B, where cells were infected with 100 VP / cell HDAd-eqIL-1Ra, horse IL-1Ra concentrations of 20–40 ng / mL were measured on days 1 and 3. When LPS was added on day 3, the IL-1Ra level increased to approximately 90 ng / mL on day 4 (group 1B), but in the absence of LPS, an IL-1Ra concentration of approximately 20 ng / mL was measured (group 1A). In groups 2A and 2B, when cells were infected with 1,000 VP / cell HDAd-eqIL-1Ra, horse IL-1Ra concentrations of 130–150 ng / mL were measured on days 1 and 3. The increase in IL-1Ra secretion after LPS stimulation was also evident in cells infected with 1,000 VP / cell HDAd-eqIL-1Ra, but the degree of induction was lower compared to cells infected with 100 VP / cell HDAd-eqIL-1Ra (Figure 14).

[0278] Regardless of LPS treatment, no significant IL-1Ra levels above baseline were detected in the HDAd-GFP-infected or vehicle-treated groups. On day 4, one repeat in group IVA (HDAd-GFP) had an IL-1Ra concentration of 53.5 ng / mL; this is considered to be a result of a technical error.

[0279] Conclusion: The results described herein indicate that cells infected with HDAd-eqIL-1Ra secreted IL-1Ra into the culture medium. IL-1Ra production by cells infected with 1,000 VP / cell HDAd-eqIL-1Ra was higher than IL-1Ra levels in the supernatant of cells infected with 100 VP / cell HDAd-eqIL-1Ra. Induction of inflammation by LPS on day 3 resulted in increased IL-1Ra levels in the supernatant of HDAd-eqIL-1R1 infected cells at 100 VP / cell and 1,000 VP / cell, and on day 4. This suggests that the inflammation-sensitive promoter system regulating IL-1Ra expression is functional.

[0280] [Example 12] Dose escalation study of helper-dependent adenovirus delivery of equine IL-1Ra in equine joints Objective of the study: This study describes a study to develop a dose of HDAd-IL-1Ra to the joints that provides high expression along with an acceptable synovial inflammatory response. HDAd-eqIL-1Ra has the same vector scaffold as FX201 or the mouse vector HDAd-mIL-1Ra, but encodes a horse-specific variant of the IL-1Ra transgene.

[0281] Methods: One 6-year-old adult horse was used for this study. The horse was examined clinically and radiographically to rule out pre-existing arthritis in the CarpJ and MCPJ of the forelimb. On day 0, the horse was administered IA injections of 3 mL dose volume of HDAd-eqIL-1Ra, formulated in PBS, into separate joints, as shown in Table 7. On day 90, the MCPJ (2 × 10⁻⁶) 11 VP (previously injected with HDAd-eqIL-1Ra), left CarpJ (6×10 12 0.125 ng of LPS was injected at a volume of 1 mL per joint into the metatarsal-phalangeal (MTPJ) joint (1 mL per joint) of the right hind limb (VP), which had been previously injected with HDAd-eqIL-1Ra. Synovial fluid from all HDAd-eqIL-1Ra-injected joints was sampled on days 0, 1, 2, 4, 7, 14, 21, 56, 90, and 92 after HDAd-eqIL-1Ra injection. In addition, synovial fluid from the right posterior MTPJ was sampled on days 56, 90, and 92, and synovial fluid from the left posterior MTPK was sampled on days 90 and 92. WBC count, protein content, and IL-1Ra levels were determined in the collected synovial fluid.

[0282] [Table 7]

[0283] Results: Clinical Signs: The results disclosed herein indicate that injection of HDAd-eqIL-1Ra into the left and right MCPJs results in moderate synovial exudation and heat on surface palpation. Twenty-four hours after HDAd-eqIL-1Ra injection, both carpal bones were swollen and warm to the touch. Exudation was severe in the left carpal bones and moderately severe in the right carpal bones. Pain that made the horse reluctant to bear weight, affecting the left forelimb and, to a lesser degree, the right forelimb, was evident over the 24 hours post-injection period. The horse preferred to remain supine for the first 24 hours after vector administration. After administering higher doses of NSAIDs for pain control over the first 36 hours, standard doses were administered until day 5. A single intramuscular dose of morphine was administered 12 hours after HDAd-eqIL-1Ra injection into all four joints. Lameness was significantly reduced on day 2, and the horse was able to walk comfortably on day 4.

[0284] Synovial fluid WBC count: The results disclosed herein show that a significant increase in synovial fluid WBC count was observed 1 day after vector injection at both dose levels. The increase in WBC count was more significant at higher vector doses. The mean total WBC count for all four injected joints was 35.2 × 10⁴ 3 The total WBC count was 1.0 × 10⁶ cells / mL; this range of total WBC counts is classified as moderate to severe inflammatory synovitis and often indicates sepsis. The difference in WBC count on day 1 showed neutrophil infiltration, which mainly changed to larger mononuclear cells by day 2. In response to NSAID treatment, the WBC count decreased to normal by day 4. The rebound in WBC count was evident on day 7, after discontinuation of NSAID treatment on day 5. Subsequently, the total WBC count gradually decreased to normal levels by day 56 and remained normal until day 90 (mean: 1.0 × 10⁶). 3 cells / mL).

[0285] Synovial fluid protein content: The disclosed results show that total protein content on day 1 post-injection was moderately elevated in MCPJ and significantly elevated in CarpJ. Synovial fluid protein content in MCPJ decreased to normal levels by day 4, but total protein content in CarpJ remained elevated until day 56. These results indicate that higher dose levels of HDAd-eqIL-1Ra induced significant synovitis.

[0286] IL-1Ra synovial fluid levels: The disclosed results show that peak synovial fluid concentrations of IL-1Ra were observed on day 1 after injection at all dose levels (Figure 15). The highest dose of HDAd-IL-1Ra resulted in a more sustained increase in IL-1Ra. A decrease in IL-1Ra levels was observed on day 4, which was considered a response to the reduction in inflammation induced by NSAID treatment. Synovial fluid concentrations of IL-1Ra increased again on day 7 after discontinuation of NSAID treatment and rebound of inflammation. By day 56, IL-1Ra was 6 × 10⁶. 12 Except for CarpJ treated with the highest dose of VP / joint, IL-1Ra was not detected in the injected joints. IL-1Ra was not detected in the synovial fluid at 90 days post-treatment. MCPJ(2×10 11 After LPS administration to VP (previously injected with HDAd-eqIL-1Ra), an increase in IL-1Ra levels was observed in the left CarpJ (6×10). 12 The increase in IL-1Ra was detected on day 92 in the right hindlimb MTPJ (previously injected with HDAd-eqIL-1Ra) and untreated right hindlimb joints. The increase in IL-1Ra was also present in the right carpJ (carp joint) that did not receive LPS. The highest reactivation of IL-1Ra expression was evident in the joints that had been previously injected with HDAd-eqIL-1Ra.

[0287] Conclusion: The disclosed results indicate that HDAd-eqIL-1Ra at full dose levels resulted in significant IL-1Ra production on day 1. The highest dose (6 × 10⁻⁶) 12VP) resulted in sustained IL-1Ra production for at least 56 days. Despite the increased IL-1Ra synthesis, higher HDAd-eqIL-1Ra doses induced more pronounced synovitis, showing transient but sustained increases in total WBC count and synovial protein content. Synovitis was associated with fairly short-term pain in CarpJs. The transient inflammatory response observed in this study may have been exacerbated by the higher total vector dose per animal, due to the treatment of four joints compared to a single joint in the efficacy study. Rebound in IL-1Ra formation at day 92 was evident after LPS injury to the joints. Levels in joints injected with HDAd-eqIL-1Ra were generally higher (ranging from 6.24 to 15.09 ng / mL) compared to naive joints injected with LPS (3.51 ng / mL). Right CarpJs not injected with LPS also showed increased IL-1Ra expression. This likely reflects local paracrine or lymphatic stimulation of CarpJs from more distal MCPJs that were inflamed by LPS injection. This result suggests that the vector may be reactivated by adjacent joint injury. In conclusion, these results indicate the potential for long-term (at least 3 months) inflammation-sensitive expression of the IL-1Ra transgene.

[0288] [Example 13] Preliminary study of helper-dependent adenovirus delivery of equine IL-1Ra for the treatment of traumatic osteoarthritis in a horse model. Study Objective: This specification describes a study to evaluate the potential of HDAd-eqIL-1Ra to treat early OA in a horse model of disease. HDAd-eqIL-1Ra has the same vector scaffold as FX201 and the mouse vector HDAd-mIL-1Ra, but encodes a horse-specific IL-1Ra transgene.

[0289] Methods: On day (-5) of the study, the midcarpal joint in one randomly selected forelimb of 12 skeletal mature (3-6 year old) thoroughbred racehorses had a surgically formed osteochondral fragment of the radiocarpal bone paired with a trendmill movement to induce early osteoarthritis (OA). Using a 10 mm curved round chisel, the fragment was separated from the dorsal surface of the radiocarpal bone, and the fracture bed was enlarged to 15 mm using an electric deburring tool. The fragment was intentionally held in the joint to induce mild degenerative changes. All horses underwent radiographic and clinical evaluation to confirm that there were no pre-existing carpal bone diseases prior to inclusion in this study. The midcarpal joint in the opposite limb was sham-operated by arthroscopy, but no fracture formation occurred. Five days after surgery (day 0), the horses were divided into experimental groups and treated with IA injection into the carpal joint that had induced OA, as shown in Table 8.

[0290] [Table 8]

[0291] Next, the horses were exercised five days a week on a rigorous program. Gait was examined, and the horses' lameness was assessed by assigning a score from 0 to 5 (0: no lameness - 5: severe lameness) using the American Association of Equine Practitioners (AAEP) scheme. To assess range of motion, the horses' legs were bent until resistance was felt, and the reduction in range was scored on a scale of 0 to 4 (0: no reduction; 1: reduction less than 25%; 2: reduction between 25% and 50%; 3: reduction between 50% and 75%; 4: reduction greater than 75%). To assess lameness after flexion, the joint was flexed for 20 seconds, and lameness was assessed using the AAEP on a scale of 0 to 5 (0: no lameness - 5: severe lameness). Finally, exudation was assessed on a scale of 0 to 4 (0: no exudation - 4: significant exudation). These clinical tests were performed weekly throughout the experiment. Furthermore, synovial fluid (cytology and IL-1Ra expression) and peripheral blood (complete blood count) were sampled on days (-5) and on days 0, 4, 7, 14, 21, 28, 56, and 72. At the end of the study (day 72), the horses were euthanized and the carpal joints were visually evaluated. Synovial and cartilage samples were processed for histological analysis and scored by pathologists in a blinded manner.

[0292] Results: Clinical Scoring: The results described herein show that horses did not develop lameness in the week following injection, indicating good tolerability of the procedure. There was also no evidence of local inflammation at the injection site after vector administration. OA joints had significantly worse exudation and range of motion scores on all days compared to sham-operated joints in weekly clinical examinations. Cumulative data regarding exudation, pain at flexion, range of motion, and lameness showed significant improvement in all tested parameters in the HDAd-eqIL-1Ra treatment group compared to horses injected with the medium (Figure 16). High-dose HDAd-eqIL-1Ra injections resulted in significant improvements in clinical parameters, but apart from lameness, the effects of higher vector doses were less pronounced than those of lower vector doses. Hematology and Clinical Chemistry: The results described herein show that no changes associated with HDAd-eqIL-1Ra vector administration were observed. Synovial Cytology: The results described herein show that low doses (2 × 10⁻⁶) were not associated with synovial cytology. 11 VP) and high dose (2 × 10 12Administration of HDAd-eqIL-1Ra to VP) induced a transient increase in WBC count in the synovial fluid compared to both untreated samples and controls injected with the medium, peaking on day 4 after injection, which resolved by day 28. Synovial fluid IL-1Ra: IL-1Ra concentrations in the synovial fluid of horses treated with HDAd-eqIL-1Ra peaked on day 4, reaching 14 ng / mL in the low-dose group and 21 ng / mL in the high-dose group. Synovial fluid IL-1Ra levels gradually decreased throughout the experiment until day 72, when they were approximately 1 ng / mL and 0.1 ng / mL in the low-dose and high-dose groups, respectively. Gross examination of injected joints: The results described herein indicate that the synovial membrane was discolored due to hemorrhage in OA joints injected with many mediums, while joints injected with HDAd-eqIL-1Ra had a normal yellowish-brown color. Untreated OA joints had significantly increased capsule thickness and abnormal discoloration. HDAd-eqIL-1Ra injection reduced both parameters to similar levels compared to sham-operated normal carpal bones. Macroscopic evaluation of the joint surface showed less fibrosis (lower scores) in the intermediate carpal bones, radiocarpal bones, the third articular surface of the intermediate carpal bones, and the third articular surface of the radiocarpal region of the joints injected with HDAd-eqIL-1Ra compared to the control joints injected with the medium. The total fibrosis score in all regions was significantly lower (better) in the HDAd-eqIL-1Ra group compared to the control group injected with the medium (Figure 17). Histological examination: The results described herein indicate that osteochondral sections from the radiocarpal bones and third carpal bones were stained with hematoxylin and eosin and examined under polarized evaluation for cartilage fibrosis, chondrocyte density, chondrocyte cloning, osteophyte formation, tidemark replication or absence, subchondral erosion, and organized structure. Further sections were examined using toluidiin blue histochemical staining to establish the regional matrix proteoglycan content, and collagen density and preservation were determined by type II collagen immunohistochemical analysis. A mean severity score was calculated in a blinded manner for each of the individual histological parameters described above, along with a total histological score combining all parameters (Figure 18).The overall histological score was significantly lower in the group treated with HDAd-eqIL-1Ra compared to the control group treated with the medium. Joints treated with HDAd-eqIL-1Ra had lower overall scores for individual parameters compared to joints treated with the medium, except for chondrocyte cloning, which was significantly reduced compared to the control group. Synovial sections from OA joints treated with the medium were thicker and showed fibrosis compared to OA joints treated with HDAd-eqIL-1Ra.

[0293] Conclusion: The results described herein indicate that surgical reconstruction of osteochondral fragments in the radial joint induces OA with moderate synovitis showing early cartilaginous changes, which manifested as significant differences in several clinical parameters, synovial cytological parameters, and histological analysis compared to sham-operated joints. Cumulative improvements in limping, range of motion, pain at flexion, and degree of exudation were evident in HDAd-eqIL-1Ra-treated joints over the course of the study. Along with overall and histological improvements in cartilage and synovium in treated joints compared to untreated OA, these results suggest that the use of HDAd vectors for direct IA delivery of the IL-1Ra transgene is safe and effective. Two HDAd-eqIL-1Ra doses, 2 × 10⁶ 11 and 2 × 10 12 VP / joint were tested, and both showed significant benefits, although one dose was not consistently more effective than the other. In conclusion, these results demonstrate a substantial benefit of IA injection of HDAd expressing equine IL-1Ra on the clinical and morphological findings of OA in a large-disease animal model.

[0294] [Example 14] Pharmacokinetics of FX201 or related species-specific constructs (HDAd-eqIL-1Ra or HDAd-rat IL-1Ra) Studies to evaluate the pharmacokinetics of FX201 or related species-specific constructs (HDAd-eqIL-1Ra or HDAd-rat IL-1Ra) are described herein. The results of the studies described herein show that FX201 or related species-specific constructs (HDAd-eqIL-1Ra or HDAd-rat IL-1Ra), administered as a single IA injection into the knee joint at doses up to 800-fold higher than the planned clinical starting dose, exhibited limited biodistribution outside the injected knee. In horses, no significant vector biodistribution was observed at the end of day 72 of the study after administration. Furthermore, no vector shed occurred throughout the study, further indicating the localization of the injected vector. In rats, vector biodistribution was primarily limited to the local injection site, including the skin, synovial lavage, quadriceps femoris, and iliac and popliteal inflow area lymph nodes, throughout the study period. Scattered, low levels of vector were observed in the liver, spleen, lungs, and bone marrow. Importantly, no vector distribution to the gonads was detected.

[0295] These results indicate that a single dose of either FX201 or HDAd-rat IL-1Ra was well tolerated in rats after ACL-T surgery. All vector-treated rats exhibited the expected immune response to the capsid, consisting of increased WBC count, T-cell response against splenic cells, swelling of localized lymph node (iliac and / or popliteal) correlating with increased lymphadenopathy, and a slight increase in arthritis and osteoarthritis. The increase in WBC count, lymphadenopathy, and lymphadenopathy had all resolved or were resolving by day 92, indicating the reversibility of this response. Despite no difference from controls at day 29, a slight increase in osteoarthritis was observed at day 92 in animals treated with both HDAd-rat IL-1Ra and FX201; this increase was thought to be primarily induced by a slight increase in arthritis.

[0296] The inflammatory response to the vector observed in the studies described herein, which may have contributed to a slight increase in the severity of osteoarthritis in the treated knee, is not expected at the low dose levels planned for human administration. Low doses (3 × 10⁶) given to a rat model of OA in efficacy studies (ACL-T 1 week prior to administration) were not expected. 7 Or 2.43 × 10 8 In VP / knee, no exacerbation of joint disease was observed; instead, administration of HDAd-rat IL-1Ra vector showed a clear therapeutic benefit against OA progression. In GLP toxicity studies, exacerbation of osteoarthritis was observed, along with increased mononuclear cell infiltration into periarticular tissue and synovial hyperplasia / hypertrophy, at a rate of 2.43 × 10⁻⁶. 10 High doses of VP / knee were observed on day 29 after treatment. However, by day 92, mononuclear infiltration and synovial hyperplasia / hypertrophy had completely resolved, and there was no clear increase in osteoarthritis. The increased inflammation of the treated joint observed in animals given FX201 at the time of the last sacrifice (day 92) can be explained by an immune response to the human IL-1Ra protein, and thus is not considered appropriate for human safety. Consistent with this, no increase in inflammation was observed on day 92 in mice treated with HDAd-rat IL-1Ra.

[0297] In conclusion, these results strongly support the idea that single local administration of FX201 or species-specific constructs is well tolerated up to approximately 1,000 times higher than the planned clinical starting dose (see Table 9). Furthermore, limited in vivo distribution outside the injected joint was observed in two species. Injection of FX201 and HDAd-rat IL-1Ra resulted in the expected non-adverse immune response to the capsid, which resolved or was in the process of resolving by the end of the study period.

[0298] [Table 9]

[0299] [Example 15] Toxicity testing of HDAd-rat IL-1Ra, a rat substitute for FX201. This specification describes a study to evaluate the toxicity and efficacy of HDAd-rat IL-1Ra, a rat surrogate for FX201, in a single-dose GLP-compliant study in a rat ACL-T model of OA. Osteoarthritis (DJD), assessed using the OARSI scoring system, was scattered in all marker knees undergoing ACL-T surgery and in some sham-surgical animals. As expected, the severity of DJD progressed from day 29 to day 92, as indicated by an increase in the central composite OARSI score for all ACL-T groups at day 92 compared to day 29, confirming the induction of OA.

[0300] All three dose levels were well tolerated, and no systemic toxicity was observed at any point. This is consistent with the limited in vivo distribution outside the injected knee after a single IA dose observed in the GLP in vivo distribution study.

[0301] The studies described herein demonstrate that the expected immune response to the Ad5 capsid was present in many animals, but both the positivity rate and magnitude were dose- and time-dependent. A dose-dependent increase in serum positivity and titer levels of circulating anti-Ad5 antibodies was observed. Titer levels were highest at day 29 compared to day 92, indicating a gradual decrease in circulating antibody levels. Furthermore, the T-cell immune response to the Ad5 vector capsid was 2.43 × 10⁶. 9 and 2.43 × 10 10 Observed in splenocytes at VP / dose. The highest dose tested was 2.43 × 10⁴. 10 In the VP / dose group, the T cell response was maintained from day 29 to day 92, but in the medium-dose group, it was 2.43 × 10⁶. 9 At VP / dose, the response had declined by day 92. At any point in time evaluated, 2.43 × 10⁻⁶ 8 At the lowest VP / dose dose, no significant T cell response to the Ad5 vector was observed.

[0302] The studies described herein did not show the systemic effects of the immune response evident in the GLP toxicity studies. At day 29 in the in vivo distribution studies, the least adverse increase in lymphadenocytes, leukocyte (WBC) count, and leukocyte percentage was observed at dose levels equivalent to the highest dose administered in the GLP toxicity studies. The absence of these findings in toxicity studies further emphasizes its non-adverse nature.

[0303] The tests described herein used the highest dose tested, 2.43 × 10⁻⁶. 10 At VP / dose, at day 29, treated ACL-T rats showed increased incidence and severity of mononuclear cell infiltration in the femorotibial joint, as well as synovial hypertrophy / hyperplasia, compared to untreated ACL-T rats and rats administered reference or low doses of HDAd-rat IL-1Ra. Mononuclear cell infiltration generally characterized some mononuclear cells in the periarticular tissue and was often associated with synovial hypertrophy / hyperplasia. This increase in mononuclear cell infiltration is thought to underlie the slight increase in OARSI scores observed at day 29 in this group compared to animals that underwent ACL-T surgery and were untreated or received reference. At day 29 in the GLP in vivo distribution study, the same 2.43 × 10⁶ values ​​were observed. 10 Animals administered VP / dose HDAd-rat IL-1Ra showed a slightly more significant local immune response to the vector compared to untreated controls. In the GLP in vivo distribution study, arthritis consisting of mononuclear or mixed cell infiltration, often associated with synovial hyperplasia / hypertrophy of the joints and adjacent tendon sheaths, was present in many animals. Importantly, and consistent with the GLP in vivo distribution study, 2.43 × 10⁶ 10 The increased incidence and severity of mononuclear cell infiltration and synovial hypertrophy / hyperplasia in animals administered VP / dose were no longer present by day 92, indicating the reversibility of these findings. In both studies, these transient effects observed in the synovium were considered non-harmful.

[0304] No further histopathological findings associated with treatment with HDAd-rat IL-1Ra were observed in the GLP toxicity studies described herein. Histopathological evaluations in the GLP in vivo distribution studies also confirmed the absence of systemic effects after treatment with HDAd-rat IL-1Ra or FX201 (excluding non-adverse cellular increases in inflow area lymph nodes).

[0305] In conclusion, the studies described herein strongly support the conclusion that a single local administration of HDAd-rat IL-1Ra, a rat species-specific construct of FX201, was well tolerated and did not result in systemic toxicity. Based on these results, the NOAEL was set at the highest dose tested, 2.43 × 10⁶. 10 The VP / dose HDAd-rat IL-1Ra is considered to be the appropriate dose. A dose scaling method based on synovial fluid volume was employed to calculate the safety margin between the NOAEL and the planned dose in the first-in-human trial. According to Emami 2018, the appropriate synovial fluid volume based on body weight in rats is in the range of 10–20 μL. Based on the body weight of the rats included in this study, 15 μL was selected for scaling between rat and human doses. As shown in Table 10, the human starting dose has a safety margin of over 800-fold relative to the NOAEL, and the maximum clinical dose has an estimated safety margin of 8.1-fold relative to the NOAEL derived from the GLP toxicity study, based on VP per ml. Importantly, since lower proposed dose levels are below the limit of quantification (BLOQ) for detection by the viral particle assay (OD260), the clinical dose would be based on GC per ml. Considering this quantitative measure, the inventors have a safety margin of 1,000 times at the starting dose and a margin of 10 times at the maximum clinical dose (see Table 11).

[0306] [Table 10]

[0307] [Table 11]

[0308] In addition to toxicological endpoints, the GLP toxicity study also included an evaluation of therapeutic efficacy. DJD, assessed using the OARSI scoring system, was scattered in all indicator knees that underwent ACL-T surgery and in some sham-surgery animals. As expected, the severity of DJD progressed from day 29 to day 92, as indicated by an increase in the central composite OARSI score for all ACL-T groups at day 92 compared to day 29, confirming the induction of OA. A moderate decrease in the central OARSI score was 2.43 × 10⁻⁶ compared to the untreated ACL-T group, rather than in the ACL-T / reference group. 9 Observations were made on day 29 in animals treated with VP / dose HDAd-rat IL-1Ra. Furthermore, on day 92, 2.43 × 10⁻⁶ was observed. 8 Animals treated with VP / dose had lower median OARSI scores compared to the untreated and reference ACL-T groups. Despite the smaller reduction in OARSI scores mentioned above, the therapeutic benefit of HDAd-rat IL-1Ra on the histopathological findings of OA was not clearly evident in this study compared to the efficacy observed in the pharmacological study in the rat ACL-T model. This can be explained by the increased disease severity at the time of treatment in this study, where HDAd-rat IL-1Ra was administered 4 weeks after ACL-T, compared to 1 week after ACL-T in the pharmacological study. It should be noted that the primary endpoint of the study was safety, and therefore, a severe disease model was selected for the GLP toxicity study in order to obtain a safety profile.

[0309] These results demonstrate that the HDAd vector's ability to express IL-1Ra in an inflammation-sensitive manner was evaluated in an in vitro study using HEK293 cells transduced with a horse-specific mutant of FX201 (Figure 6). Stimulation of NF-κB signaling with lipopolysaccharide (LPS) resulted in increased horse IL-1Ra expression compared to unstimulated controls, confirming the function of the NF-κB-inducible promoter.

[0310] These results confirm the function of the HDAd vector system in vivo in a dose-escalation study conducted in a single healthy horse. The study involved four dose levels (2 × 10⁶) of HDAd-eqIL-1Ra, a horse variant of FX201. 11 ~6×10 12 The VP / dose was administered to separate joints in horses (left and right carpal joints (CarpJ) and left and right metacarpophalangeal joints (MCPJ)). The results indicate robust IL-1Ra expression in the synovial fluid was observed 1 day after administration at all dose levels. Injection of the vector in this study was associated with transient motor impairment due to arthritis, and increased leukocyte (WBC) count and total protein levels in the synovial fluid. Since no local intolerance was observed in other studies, the transient inflammatory response observed in these results is significant compared to a single joint in efficacy studies (highest dose tested was 2 × 10⁶). 12 (This was due to the high total vector dose per animal resulting from the treatment of four joints (8.8 × 10 in a single animal). 12 It is presumed that the condition worsened due to total exposure to VP.

[0311] IL-1Ra levels decreased in response to systemic nonsteroidal anti-inflammatory drug (NSAID) administration on days 2–5, and a rebound in expression was observed after discontinuation of NSAIDs. IL-1Ra was detectable in synovial fluid 56 days after injection. Increased IL-1Ra expression was induced by injection of LPS into previously vector-treated joints 90 days after treatment. Therefore, these results suggest the potential for long-term (at least 3 months, total length of the study) inflammation-susceptibility expression of the IL-1Ra transgene.

[0312] The results disclosed herein are 2 × 10 in both pain and functional parameters, as well as in the preservation of joint structure. 11 or 2 × 10 12This study demonstrates the efficacy of HDAd-eqIL-1Ra at VP / dose levels in an equine osteochondral fragment model. Both dose levels of HDAd-eqIL-1Ra resulted in reduced lameness, lameness after joint flexion, reduced joint exudation, and improved range of motion 72 days after administration. Furthermore, both dose levels of HDAd-eqIL-1Ra resulted in structural improvements in the injured joint compared to the medium-treated animals at the time of the study's conclusions. These included overall reductions in macroscopic and microscopic joint findings in the cartilage, as well as improvements in synovial surface color and thickness.

[0313] The result this time is 3 x 10 7 and 2.43 × 10 8 Further confirmation of OA in a rat anterior cruciate ligament transection (ACL-T) model with VP / dose HDAd-rat IL-1Ra. Animals received a single IA injection of HDAd-rat IL-1Ra one week after ACL-T surgery, and the joint was evaluated for histopathology of cartilage / bone and synovial tissue 11 weeks later. Our results disclose that animals undergoing ACL-T surgery developed microscopic changes associated with OA in the knee joint. Our results disclose that HDAd-rat IL-1Ra dose-dependently reduced the incidence and severity of these changes in both cartilage / bone and synovial tissue, demonstrating the therapeutic benefit of this treatment for structural progression of OA.

[0314] In a more severe model of OA, the efficacy of HDAd-rat IL-1Ra was observed, and it was given 2.43 × 10⁻⁶ times four weeks after ACL-T surgery. 8 Or 2.43 × 10 9 A single IV injection of VP / dose reduced the central composite OARSI score for cartilage / bone one month after administration (at day 29). The lower dose was 2.43 × 10⁻¹⁰. 8 VP / dose also resulted in a reduction in OARSI scores at 3 months after administration (at day 92) compared to ACL-T untreated or media-treated (reference) animals.

[0315] In summary, the results from the nonclinical pharmacological studies described herein demonstrate the function of the HDAd-IL-1Ra vector in delivering therapeutic levels of IL-1Ra in the presence of inflammation. Furthermore, a single dose of HDAd-IL-1Ra resulted in reduction of pain and loss of function in horses, as well as symptoms such as OA of structural joint injury in mice, rats, and horses.

[0316] For clinics, FX201 allows for vector quantification using Droplet Digital® polymerase chain reaction (ddPCR). Table 12 below establishes how the minimum effective nonclinical dose relates to our target clinical dose.

[0317] [Table 12]

[0318] These results demonstrate that, after IA injection, FX201 infects cells in the joint and locally produces IL-1Ra in response to inflammation. The results described herein show that the HDAd vector provides a desirable profile for maintaining a sustained therapeutic level of IL-1Ra locally in the injected joint. This is evidenced by the robust expression patterns of marker genes (GFP and LacZ) observed after a single injection into mouse joints, demonstrating favorable expression patterns in the superficial cells of synovial and chondrocytes. Transgene expression after delivery by HDAd was sustained, continuing for 378 days after a single IA injection in normal mouse joints, a timeframe limited by the animal's lifespan, and transgene expression did not gradually decline. Furthermore, quantifiable levels of IL-1Ra were present in synovial fluid derived from horses 72 days after vector administration, further supporting the persistence of transgene expression.

[0319] The efficacy observed after HDAd-IL-1Ra administration throughout nonclinical efficacy studies (in mice, rats, and horses) indicates that effective levels of IL-1Ra are achievable in the joints for up to 3 months after administration, over the duration of the study period. The range of dose levels was tested across five efficacy studies. A synovial fluid volume-based scaling method was employed to allow comparison of effective dose levels among mouse, rat, and horse OA models (Table 13). The minimum effective dose, based on studies in rat ACL-T, was 2 × 10⁶ per mL of synovial fluid. 9 It was defined as VP.

[0320] [Table 13]

[0321] These results indicate that HDAd-IL-1Ra treatment resulted in reduced pain, functional impairment, and structural progression. Improvements in structural progression included reductions in cartilage fibrosis and tear formation, chondrocyte death and cloning, subchondral bone remodeling, and osteophyte proliferation. Importantly, these beneficial effects on joint structure were observed when HDAd-IL-1Ra was administered at various time points associated with joint injury (from before injury to 4 weeks after injury).

[0322] In summary, nonclinical pharmacological studies conducted using FX201 (HDAd-IL-1Ra) disclosed herein demonstrated the ability to deliver sustained locally transgeneized expression sufficient to provide robust efficacy in reducing pain, functional impairment, and structural progression of joint changes such as OA in both small and large animal models of OA. The results of the studies described herein strongly support and provide a basis for the use of effective doses of HDAd encoding IL-1RA of the present invention for the treatment and prevention of osteoarthritis in humans suffering from osteoarthritis or an osteoarthritis-like condition.

[0323] [Example 16] Clinical trial design to evaluate the safety and efficacy of IL-1Ra gene therapy (FX201, humantakinogene hadenovec) for IA administration developed for the treatment of patients with osteoarthritis of the knee (OAK). Patient and test procedures This specification describes an open-label, single-dose escalation study to evaluate the safety and tolerability of FX201 in patients with osteoarthritis of the knee (OAK). The primary objective of the study described herein is to evaluate the safety and tolerability of a single IA injection of FX201 in patients with OAK, as measured by adverse events (AEs) spontaneously reported by patients or discovered by the investigator, as well as by the following assessments: findings from physical examination, assessment of the benchmark knee, vital signs, electrocardiogram (ECG), and laboratory evaluation. Furthermore, the benchmark knee will be evaluated by radiography and assessed by a central imaging provider for chondrolysis, subchondral changes, osteonecrosis, and incomplete fractures.

[0324] All patients receive a low dose (2.8 × 10⁻¹⁰). 9 GC / ml), medium dose (2.8×10 10 GC / ml) or high dose (2.8 × 10) 11 Patients receive a single IA injection of FX201 at GC / ml. Patients are assigned doses by cohort, starting with the lowest dose and increasing. Up to three escalating doses of FX201 will be tested in cohorts of 5-8 patients. Each patient receives a single injection of FX201 and is followed for 104 weeks. The trial will be conducted in male and female patients aged 30-80 years with indicative knee pain-heavy OA with Kellgren-Lawrence (KL) grade 2, 3, or 4 (KL grade 4 for intermediate and high-dose cohorts only). After informed consent, patients meeting the eligibility criteria will be selected for enrollment. Up to three escalating doses of FX201 will be tested in cohorts of 5-8 patients. A total of up to 24 patients will be recruited for the trial. Each patient receives a single injection of FX201 and is followed for 104 weeks.

[0325] Adverse events are graded according to their severity in accordance with the Common Terminology Criteria for Adverse Events (CTCAE) v5.0. If the severity of an AE cannot be specifically graded, the principal investigator should use medical judgment to apply the general guidelines for determining grades 1 through 5 listed in CTCAE v5.0. Details of the dose escalation protocol are as follows: a) Low-dose cohort (Dose A - 2.8 × 10 9 GC / ml): b) Medium dose (dose B-2.8×10 10 GC / ml): and c) High-dose cohort (Dose C - 2.8 × 10 11 GC / ml).

[0326] Each cohort includes 5 to 8 patients. The first patient is treated and monitored for 7 days to assess safety. If no grade 3 or higher AEs associated with or considered to be related to the study drug or the study injection procedure are observed within 7 days post-treatment, the second patient is treated and monitored. If no grade 3 or higher AEs associated with or considered to be related to the study drug or the study injection procedure are observed within 7 days post-treatment, three additional patients are enrolled and followed for at least 4 weeks. If no grade 3 or higher AEs associated with or considered to be related to the study drug or the study injection procedure occur in the first five patients, the data are reviewed and further recommendations are considered. If a grade 3 or higher AE associated with or considered to be related to the study drug or the study injection procedure occurs in any of the first five patients, three additional patients are enrolled in the cohort. If a Grade 3 or higher AE occurs that is related to, or likely to be related to, the investigational drug or the investigational injection procedure, patient enrollment will be suspended until data is reviewed and a recommendation is made to continue the protocol as planned, modify the protocol, or discontinue enrollment. If a serious adverse event (SAE) occurs at any point in time, regardless of its relevance, patient enrollment will be suspended and data will be reviewed to provide a recommendation to continue the protocol as planned, modify the protocol, or discontinue enrollment. For the last patient in the cohort, safety data for all patients will be reviewed for at least 4 weeks after administration to determine whether a) an increase to the next dose level to proceed to the next dose trial occurs for the low-dose and medium-dose cohorts; or b) in the case of the high-dose cohort, to determine the level for future trials.

[0327] Selection of test group Number of patients. A maximum of 24 patients (ranging from 15 to 24) are enrolled and treated with a single IA injection of FX201.

[0328] Study patient eligibility criteria To be included in the clinical trial, patients must meet the following criteria: 1. A consent form to participate in the trial; 2. The willingness and ability to comply with the trial procedures and visit schedule, and the ability to follow verbal and written instructions; 3. A male or female between 30 and 80 years of age (inclusive) on the registration day (Day 1); 4. A weight of 40 kilograms or less per square meter (kg / m²) at the time of screening. 2) Body Mass Index (BMI); 5. Indicative knee OA-associated symptoms for at least 12 months prior to screening (patient-reported symptoms are acceptable); 6. Indicative knee pain for more than 15 days in the last month prior to screening (patient-reported symptoms are acceptable); 7. American College of Rheumatology (ACR) criteria for OA (clinical and radiographic) (Altman et al, 1986): a) knee pain, b) at least one of the following: age over 50, morning stiffness of less than 30 minutes, friction sounds when moving the knee, and c) osteophytes; 8. Conservative treatment for two or more indicators of osteoarthritis of the knee (e.g., a planned athletic exercise program (strengthening and / or aerobic exercise and / or balance training / neuromuscular exercise and / or mind-body exercise including Tai Chi or yoga)); topical nonsteroidal anti-inflammatory drugs (NSAIDs); failure of non-selective NSAIDs or COX-2 inhibitors; or one of the aforementioned conservative treatments and at least one of the aforementioned indicators of knee IA treatment (corticosteroids or hyaluronic acid) (Bannuru RR et al, 2019) Failure; 9. Kellgren-Lawrence (KL) grade 2, 3, or 4 in an indicator knee based on X-rays performed during screening and confirmed by a radiographer trained at the central facility prior to enrollment (KL grade 4 for cohorts B and C only) (Grade 2: obvious osteophytes and possible joint space narrowing, Grade 3: moderate multiple osteophytes, obvious joint space narrowing and possible some sclerosis and deformity of the epiphysis, and Grade 4: large osteophytes, significant narrowing, severe sclerosis and obvious deformity of the epiphysis); 10. Indicative knee examination showing that the indicator knee and intended injection site area do not contain any signs of local or joint infection at baseline; 11. Western Ontario and McMaster Universities Osteoarthritis in an indicator knee with a numerical rating of ≥4.0 and ≤9.0 (0-10 scale [NRS]) at screening visit and baseline. 12. The registration slot is available within the 21-day screening period confirmed by the sponsor; 13.If confirmed by the sponsor, registration slots are available for patients with KL grade 4 in the knee, which is an index based on central readings of X-ray screening (maximum of 2 KL grade 4 patients per cohort in cohorts B and C only); 14. Sexually active women of childbearing potential (defined as not surgically infertile or postmenopausal [defined as no menstruation for 12 consecutive months without another medical cause documented in medical history]) must have a negative serum pregnancy test at baseline (before registration) and agree to use one of the following highly effective methods of contraception: abstinence; oral, injectable or implantable hormonal contraception; intrauterine devices or systems; condoms or closure caps (pessaries or cervical / vaginal fornix caps) with spermicide bubbles / gels / films / creams / suppositories; or monogamous intercourse with a surgically infertile partner (must be 6 months post-vasectomy) for at least 12 months after administration of the study drug. Sexually active men must agree to use condoms during sexual contact with women for at least 12 months after receiving the study drug.

[0329] During the trial, document and report all existing (started before the start of the trial), new concomitant medications, or any changes thereto, and the relevant reasons for their use or modification.

[0330] Exclusion criteria Disease-related criteria: 1. Current or previous diagnosis of reactive arthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, or arthritis associated with inflammatory bowel disease; 2. Current or previous history of infection in a benchmark knee joint; 3. Clinical signs and symptoms of active crystalline disease of the benchmark knee (gout, calcium pyrophosphate deposition) within 3 months of screening; 4. Inability to undergo magnetic resonance imaging (MRI) due to surgical hardware or other foreign bodies in the benchmark knee; 5. Unstable benchmark knee joint (e.g., anterior cruciate ligament injury) within 12 months of screening.

[0331] Criteria related to previous or concomitant treatments: 1. Use of any IA drug / biopharmaceutical in the knee within 6 months of screening (e.g., corticosteroids, hyaluronic acid, platelet-rich plasma, stem cells, prolotherapy, and amniotic fluid injection); 2. Cryo- or radiofrequency nerve ablation of the knee within 12 months of screening; 3. Arthroscopic or open surgery of the knee within 12 months of screening.

[0332] Patient-related criteria: 1. Women who become pregnant, breastfeed, or plan to become pregnant within 12 months of administration; men who plan to become pregnant within 12 months of administration; 2. Loss of skin integrity on the knee as an indicator for injection; 3. Prediction of major surgery within 12 months of administration; 4. Clinical laboratory values ​​for human immunodeficiency virus (HIV) infection, positive test for hepatitis B surface antigen (HBsAg), or hepatitis C virus (HCV) ribonucleic acid (HCV) 1. Positive test for RNA and serological positivity for hepatitis C virus; 5. ECG abnormalities at screening or baseline visit determined to be clinically significant by the principal investigator or delegate; 6. Use of immunomodulators, immunosuppressants, or chemotherapeutic agents within 5 years prior to screening; 7. Previous clinical trial or approved gene therapy treatment; 8. Active malignancy or history of malignancy within the last 5 years, excluding excised basal cell carcinoma, squamous cell carcinoma of the skin, or effectively controlled cervical intraepithelial neoplasia; 9. Aggressive pharmacological treatment for depression, including selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), non-selective serotonin reuptake inhibitors (NSRIs), or tricyclics, if the dose / regime has not been stable for more than 6 months prior to screening; 10. Past 12 months 11. Active substance abuse (drugs or alcohol) within the past three months or a history of substance abuse; 12. Use of other investigational drugs, biopharmaceuticals or devices within three months prior to screening; 13. Systemic or local bacterial or viral infection requiring IV antibiotics or antivirals within four weeks prior to screening or oral antibiotics or antivirals within two weeks prior to screening; 14. If bilateral knee OA is present, pain in the opposite knee of 4.0 or higher (on an NRS scale of 0-10) within one month prior to the screening visit; 15. Previous total or partial knee arthroplasty in the index knee; 16. Temperature above approximately 99.5°F (37.5°C) at baseline; 17. Prothrombin time (PT) / International Normalized Ratio (INR) greater than 1.5; activated partial thromboplastin time (aPTT) greater than 5 seconds above the upper limit of normal (ULN); 1. ULNAlanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels greater than 5 times; alkaline phosphatase (ALP) levels greater than 1.5 times ULN; total bilirubin levels outside the normal range; 18. known allergies or sensitivity to acetaminophen; and 19. any other clinically significant acute or chronic medical condition (e.g., bleeding disorder) that, in the judgment of the principal investigator, could exclude the use of IA injection or impair patient safety, limit the patient's ability to complete the study, and / or undermine the purpose of the study.

[0333] Investigational drug administration procedure FX201: humantakinogene hadenovec; low, medium, and high doses are administered as a single 5 mL IA injection. Concentrations of three formulations: dose level A: 2.8E9 GC / mL (genome copy number per milliliter); dose level B: 2.8E10 GC / mL; and dose level C: 2.8E11 GC / mL.

[0334] As part of eligibility, the reference knee should be examined at baseline to ensure that the knee and the intended injection site area do not show any signs of local or joint infection. Intra-articular injection of the study drug will be performed by an assigned injector. The injector may select the knee position to be used (e.g., extended or flexed), the technique for injection (e.g., central or lateral), and the anesthetic (e.g., EMLA cream, subcutaneous lidocaine 1%) based on standard treatment. Aseptic techniques should be used. Prior to injection, the reference knee should be thoroughly washed with a bactericidal solution. In all cases, the reference knee should be aspirated prior to administration of the study drug to collect synovial fluid for IL-1Ra and IL-1β protein concentrations.

[0335] Synovial fluid can be collected using mechanical compression. If IA exudate is detected using ultrasound guidance, the person administering the injection should withdraw the fluid until it is nearly dry before injection. After attempting synovial fluid aspiration, inject 5 mL of FX201 into the synovial cavity using ultrasound guidance. After aspiration and injection, pressure should be applied to the injection site using sterile gauze. The injection site should be wiped with alcohol and covered with an adhesive pad and bandage. All injection / aspiration equipment should be disposed of in accordance with local organization procedures for the disposal of biohazardous materials.

[0336] The same needle used for synovial fluid aspiration may be used for the IA injection of the test drug, thereby allowing for a single injection with a syringe change. The injector will use a 21 gauge or larger needle for synovial fluid injection and aspiration. The injector will record any problems with the injection procedure and administration of FX201 and report any incidents to the site monitor.

[0337] Previous medications and concomitant medications Acceptable drug / non-pharmacological therapies Throughout the trial, the following medications and non-pharmacological therapies may be taken or used: a) any treatment for AEs; b) emergency medications assigned to the trial; c) ice applied locally to rest, elevate or indicator knees; d) any treatment for pre-existing conditions other than those indicated for the trial, which are limited and not listed; e) aspirin for cardioprotection at the maximum dose of 81 mg per day, provided the dose has been stable for more than three months prior to entering the trial; f) pharmacotherapy for depression, including SSRIs, SNRIs and NSRIs or tricyclics, provided the dose and regimen have been stable for six months prior to screening; g) Patients should be advised to maintain a stable lifestyle with respect to physical activity, physiotherapy, acupuncture, TENS or braces throughout the 52-week treatment period after medication.

[0338] Prohibited drugs / non-pharmacological therapies The following medications and non-pharmacological therapies should not be taken or administered after the patient has signed informed consent, or used for a 52-week treatment period: a) oral NSAIDs; b) topical therapies applied to the indicator knee (e.g., topical NSAIDs, capsaicin, lidocaine patches, other topical treatments); c) cannabinoids; d) aspirin (more than 325 mg per day); e) centrally acting analgesics (e.g., pregabalin, gabapentin); f) opioids (oxyco) (g) Dong, hydrocodone, codeine, morphine, tramadol, etc.; (h) Muscle relaxants (e.g., cyclobenzaprine, tetrazepam, diazepam); (i) Any IA injections in the index knee (e.g., local anesthetics, corticosteroids, hyaluronic acid, platelet-rich plasma, stem cells, prolotherapy, amniotic fluid injection); (i) Cryo- or radiofrequency nerve ablation in the index knee; (j) Any investigational drug, device, or biopharmaceutical; (k) Any immunomodulator, immunosuppressant, or chemotherapeutic agent.

[0339] Oral NSAIDs, topical therapies, and topical cannabinoids applied to the benchmark knee are prohibited by informed consent for up to 60 days post-treatment. After 60 days post-treatment, their use is restricted at the discretion of the principal investigator. A washout (at least 5 half-lives) must be completed at least 10 days prior to the baseline visit.

[0340] Restricted drugs After the first 60 days following the treatment, the use of the following medications may be restricted at the discretion of the principal investigator based on the clinical need for additional pain relief if the designated emergency medications are insufficient. However, patients should not take these medications within 72 hours prior to a study visit during the 52-week treatment period after administration: oral NSAIDs, topical therapies applied to the indicator knee (e.g., topical NSAIDs, capsaicin, lidocaine patches, other topical treatments), and topical cannabinoids.

[0341] First aid To standardize pain relief emergency medication across all patients, it will be initiated at screening (after informed consent), and patients will discontinue all prohibited medications. The designated emergency medication is acetaminophen 500 mg. Patients will be instructed to take 1-2 tablets every 6 hours as needed, and not to exceed 6 tablets (3000 mg) in 24 hours. Patients will be provided with the emergency medication starting at screening and throughout the 52-week treatment period. If instructed, patients will return the emergency medication for emergency medication reporting purposes and receive a new supply. Patients will be provided with a sufficient amount of emergency medication until the first visit, from day 1 to the first week visit, and for subsequent visits up to week 52. Patients will record the use of the emergency medication from screening to 60 days post-administration for Cohort A, and from screening to 84 days post-administration (12 weeks) for Cohorts B and C. No emergency medication will be provided during the long-term follow-up period (visits from week 52 to week 104).

[0342] Test variables Safety variables: Safety and tolerability are assessed based on eugenics (AEs) spontaneously reported by patients or discovered by the principal investigator, as well as the following assessments: findings from physical examination, benchmark knee assessment, vital signs, ECG, and laboratory evaluations. The benchmark knee is assessed by radiography and evaluated by a central imaging provider for chondrolysis, subchondral changes, osteonecrosis, and incomplete fractures.

[0343] Efficacy Variables: Efficacy is assessed independently based on the WOMAC 3.1 pain subscale (0-10 NRS scale) and WOMAC 3.1 rigidity subscale (0-10 NRS scale), as well as KOOS pain, other symptoms, function in activities of daily living (ADL) (0-100 NRS scale), and sports and recreational function and knee-related quality of life (QoL) (5-point Likert scale). The indicator knee is evaluated by MRI, and quantitative changes in bone morphology are assessed by a central imaging provider.

[0344] In vivo distribution variables: Plasma exposure is analyzed from plasma samples. Shedding is analyzed from urine samples and skin swabs at injection sites.

[0345] Bioanalytical variables: The presence of anti-capsid antibodies and anti-IL-1Ra antibodies will be analyzed from blood samples. High-sensitivity C-reactive protein (hs-CRP) levels will be analyzed from blood samples. IL-1Ra and IL-1β protein concentrations will be analyzed from synovial fluid samples. Synovial fluid and blood samples will be stored for up to 5 years after the completion of the study for potential future analysis of biomarkers that may contribute to the development of OA and / or be associated with responsiveness to FX201 treatment. No genomic analysis (gene sequencing) will be performed using these samples. Patients can withdraw their consent at any time during the storage period. Once analysis has begun, consent can no longer be withdrawn.

[0346] Statistical methods: Four analytical populations are planned for the trial. The safety population includes all patients receiving the study drug. The safety population is used to assess safety and tolerability. The biodistribution population includes patients receiving the study drug and having at least one post-treatment biodistribution sample. The biodistribution population is used to assess the biodistribution of FX201 vector copies. The bioanalysis population includes patients receiving the study drug and having at least one post-treatment bioanalysis sample. The bioanalysis population is used to assess the bioanalysis effect of FX201. The full analysis set (FAS) population includes all patients receiving a full dose of the study drug and having baseline and at least one post-administration evaluation. The FAS population is used to assess preliminary efficacy endpoints.

[0347] Safety analyses are performed on the safety population. Adverse events are coded using the Medical Dictionary for Regulatory Activities (MedDRA) dictionary and graded for severity according to CTCAEv5.0. The incidence (number and percentage) of treatment-related adverse events (TEAEs), which are events that started after administration or worsened in severity after administration, is presented by dose group. The incidence of TEAEs is also presented by maximum severity and relationship to the study drug. Similar presentations are provided for single-acting adverse events (SAEs), AEs resulting in death, AEs resulting in withdrawal from the study, and index knee-related AEs. Clinical laboratory values, ECGs, vital sign information, and radiographic screening data are summarized as summary statistics for values ​​at individual time points and changes from baseline, respectively. Summary statistics include n, mean, median, standard deviation (SD), minimum, and maximum. Categorical variables are summarized using frequency and percentage.

[0348] CTCAEs use a unique clinical description of the severity of each AE, based on these general guidelines, to indicate grades 1 through 5: Grade 1: Asymptomatic or mild symptoms; clinical or diagnostic observation only; no intervention required; Grade 2: Requires minimal, local or non-invasive intervention, or age-appropriate limitation of instrumental ADL; Grade 3: Severe or medically significant, but not immediately life-threatening; requires hospitalization or extended hospitalization; functional impairment; limitation of daily living ADL; Grade 4: Life-threatening consequences or requires emergency intervention; and Grade 5: Death associated with the AE.

[0349] The percentage of patients positive for FX201 at each time point is presented by dose group. The observed vector copy number of FX201 is summarized by dose group and sample type at each biodistribution time point. The ratio of IL-1Ra to IL-1β is reported. Baseline plasma hs-CRP levels are reported. Antidrug antibodies against adenovirus serotype 5 (Ad5) capsid and IL-1Ra are reported. In addition, neutralizing antibody titers are reported for each Ad5 seroconverted patient.

[0350] The preliminary efficacy data collected in this study are presented using summary tables, diagrams, and a list of data points. The summary tables present the data by dose group and, where applicable, by time of collection. Continuous variables are summarized using descriptive statistics, specifically the mean, median, standard deviation, minimum, and maximum values.

[0351] Categorical variables are summarized using frequency and percentage. Confidence intervals may also be provided. Figures are used to assist in the presentation of specific data. Sensitivity analysis can be performed to examine the effects of missing data, as well as any baseline imbalances. All CIs, statistical tests, and resulting p-values ​​are provided for informational purposes only and are reported as two-sided. Significance is assessed at the α=0.05 level, and the significance level is not adjusted for multiplicity.

[0352] Initial observation - Preliminary data from the low-dose cohort of the clinical trial. Following administration of the lowest dose of FX201 (FX201-2019-001; NCT04119687), the treatment responses observed to date in initial clinical trials of FX201 strongly support the potential of FX201 treatment to benefit patients with osteoarthritis pain. Individual patient responses, measured by the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) A (pain), were evaluated using thresholds defined by the Initiative in Methods, Measurements and Pain Assessment in Clinical Trials (IMMPACT) group for clinically significant improvement in the outcome of pain treatment. In this cohort of patients with moderate to severe osteoarthritis pain at baseline, three of the five treated patients (60%) who reported a decrease in their WOMAC-A pain score were measured for substantial improvement (greater than 50% improvement from day 1) at 8 weeks post-treatment, meeting the IMMPACT criteria.

[0353] Low doses of FX201 (1.4 moles x 10¹⁶) were administered to patients tested in the initial stages. 10 2.8 × 10⁻¹⁶ per total GC volume 9 Considering the GC (Critical Care), the improvements observed in the patients with moderate to severe osteoarthritis described above are truly remarkable and unexpected.

[0354] [Example 17] Specifications of FX201 formulation Methods for evaluating the quality of FX201 formulations, including the adenovirus-based biological delivery and expression systems described herein, along with acceptance criteria, are described herein.

[0355] Appearance: Visual observation

[0356] Pass / fail criteria: The solution must be transparent to slightly milky white, colorless, and free of visible particles.

[0357] Visual evaluation is a standard technique for assessing appearance. The FX201 formulation contains a colorless excipient and may appear slightly milky white due to the concentration of virus particles in the formulation.

[0358] Identity of target gene sequences: DNA sequencing

[0359] Pass / fail criteria: The IL-1Ra sequence matches the reference sequence.

[0360] The purpose of this method is to accurately identify FX201. The DNA sequence obtained by Sanger sequencing is compared to a human IL-1Ra reference sequence. The sequenced IL-1Ra must match the reference sequence to ensure that the formulation contains the target gene.

[0361] Viral vector identity: Droplet Digital polymerase chain reaction (ddPCR)

[0362] Pass / fail criteria: Detection of helper-dependent adenovirus (HDAd)

[0363] The FX201 HDAd viral genome copy (GC) count is measured by ddPCR using FX201-specific primers that target unique regions of the genome. Because the method is specific to the FX201 HDAd genome, its ability to detect the HDAd genome confirms the identity of the viral vector.

[0364] Chemical / physical properties

[0365] pH:

[0366] Pass / fail criteria: 7.0 ± 1.0

[0367] Potentiometric measurement is a technique of choice based on accuracy and is the industry standard. pH is measured using USP <791> Testing using Ph.Eur.2.2.3 ensures the stability of the FX201 formulation and its physiological compatibility with intra-articular administration.

[0368] Particle size / aggregation: Dynamic light scattering (DLS)

[0369] Cumulant: Hd and Pd (%)

[0370] Normalized main peaks: Hd, intensity (%), and Pd (%)

[0371] The purpose of this method is to measure the size distribution of viral particles present in the FX201 formulation using a dynamic light scattering instrument. The level of aggregated viral particles is monitored because aggregation can lead to decreased potency and increased immunogenicity. The reported results include particle size, standard deviation, and mean cumulant peak polydispersity and all normalized major peaks.

[0372] Osmolality: USP <785>

[0373] Pass / fail criteria: 600 mOsm / kg or less

[0374] Osmolality is USP <785> The osmolality is determined using an official method. The selected freezing point depression method is an industry standard method. The osmolality of the injection solution may affect the product's acceptability or physiological compatibility. A specification limit of 600 mOsm / kg or less is established to ensure the acceptability of FX201 injection (Roethlisberger 2017). The osmolality of FX201 is primarily driven by a formulation buffer (approximately 450 mOsm / kg) with a specification of 600 mOsm / kg or less, establishing an upper limit for the final formulation. The range for each dose level represents an acceptable 2x error around the target concentration of 2.8E+X at each dose level. The range is suitable for this development stage because the target dose levels for the three doses represent a 10x difference.

[0375] Infectivity assay: 50% tissue culture infectious dose (TCID) 50 )

[0376] As described herein, the objective of this method is to determine the infectivity titer of the FX201 formulation in HEK293 cells. FX201 is HDAd, a non-replicating recombinant viral vector containing a human IL-1Ra sequence. HDAd requires adenovirus 5 (Ad5) virus as a helper for replication. TCID 50 The assay is performed in HEK293 cells, during which the cells are co-infected with serial dilutions of the FX201 formulation sample and saturated Ad5. After incubation, the amplified virus is detected by qPCR targeting the unique sequence of the recombinant HDAd vector. Based on the qPCR results, wells inoculated with serial dilutions of the HDAd sample are scored for the presence or absence of the virus, and a TCID (Tripartite Certification Index) is assigned. 50 (TCID 50 The ratio () / mL) is calculated using the Spearman-Karber method.

[0377] Ratio of virus particles to infection units: Calculation

[0378] Pass / Fail Criteria: 300 GC / TCID 50 below

[0379] This is TCID, which represents the number of virus particles expressed as GC per 1 ml. 50 This is a calculation of the ratio to the infectious unit expressed as / mL. In the current development stage, the maximum viral particle infectious unit ratio is 400 GC / TCID. 50 The following settings define the maximum acceptable limits, while allowing for further batch experience and methodological refinement. This pass / fail criterion is based on the batch history data collected to date, taking into account the accuracy of the collected assays used to determine the ratios. Continue collecting batch data and refine the method during program development.

[0380] TCID 50This is a measurement of the number of infectious units per 1 mL of pure FX201 formulation. An infectious unit is a single infection event, which is a measurement that determines the infectivity of a virus particle. In an event where the virus particles in an FX201 batch have the same infectivity, the change in the concentration of virus particles in the sample is TCID. 50 This can affect the readings. At the same time, if FX201 batches had different infectivity but similar amounts of viral particles, different TCIDs may be obtained for each batch. 50 A value may exist. Therefore, the infectivity of a virus particle is ultimately determined by the ratio of the virus particle to the infectious unit.

[0381] IL-1Ra expression assay: Enzyme-linked immunosorbent assay (ELISA)

[0382] Pass / fail criteria: IL-1Ra is detected.

[0383] HEK293 cells that do not express IL-1Ra are infected with FX201. After incubation, the cell supernatant is collected, concentrated, and analyzed for the presence of IL-1Ra. A commercially available sandwich ELISA kit is used to detect human IL-1Ra expression.

[0384] The cellular expression of IL-1Ra after FX201 transfection requires the presence of an inflammatory promoter that results in variable and transient production of IL-1Ra. Due to the potential of variable expression and its early stages of development, the current pass / fail criterion is the detection of IL-1Ra expression. This method has been adequately qualified to demonstrate its ability to detect IL-1Ra expression in HEK293 cells infected with FX201.

[0385] Sterility according to ICH Q4B Annex 8 (R1)

[0386] Pass / fail criteria: Sterile / No proliferation

[0387] Sterility was tested using a sterility test, specifically a direct inoculation method on the final vial of the FX201 formulation, and the required number of samples were USP <71> Defined in / Ph.Eur.2.6.1 / JP4.06. Three official methods have been harmonized under ICH Q4B Annex 8(R1), and product-specific qualification has demonstrated that the official methods are compatible with the FX201 formulation. As a sterile formulation, the FX201 formulation shall not show bacterial / fungal growth.

[0388] endotoxins

[0389] Pass / fail criteria: 35 EU / mL or less

[0390] USP <85> A bacterial endotoxin assay is performed according to the specified procedure to quantify the level of Gram-negative bacterial endotoxins in the FX201 formulation.

[0391] FX201 is administered in 5 mL doses. The endotoxin limit of 35 EU / mL corresponds to 175 EU / dose. This is equivalent to 2.5 EU / kg for an average adult weighing 70 kg, within the parent formulation guidelines of 5 EU / kg or less.

[0392] The method for producing adenovirus-based biological delivery and expression systems disclosed herein is based on the method for large-scale production of high-quality helper-dependent adenovirus vectors using adherent cells in a cell factory, as described in Suzuki et al., HUMAN GENE THERAPY 21:120-126 (January 2010).

[0393] [Example 18] Manufacturing process overview The manufacturing process described herein is based on adherent cell proliferation described by Suzuki, 2010, using CellStacks® (CS). 116 cell lines derived from HEK293 cells are co-infected with FX201 and a helper virus bank. After co-infection, the cells are harvested and lysed. The amplified FX201 is purified by three rounds of cesium chloride (CsCl) gradient ultracentrifugation, with the band containing enriched FX201 being extracted for further processing during each ultracentrifugation cycle. The resulting purified FX201 in CsCl is dialyzed with a formulation buffer to remove CsCl, then diluted to the target concentration, filtered, and packed. The resulting FX201 formulation material is frozen and stored at -65°C or below and tested for release.

[0394] Upstream process Cell culture process A cell culture process beginning with thawing vials of 116 master cell bank (MCB) is described herein. The thawed cells are enlarged and co-infected with FX201 and helper virus bank. After infection, the cells are supplied and harvested, and the cell culture process is completed before further harvesting for purification.

[0395] Thawing vials: Thaw 116MCB vials in a 37°C water bath. Cell viability at thawing should be 70% or higher. Remove the preservative (dimethyl sulfoxide) from the vials, place the cells in a culture flask containing the culture medium, and incubate in a 5% CO2 incubator at 37°C until the cells reach a sufficient density for the next cell expansion step. The culture medium used for vial thawing and initial passage consists of Dulbecco's Modified Eagle Medium (DMEM) supplemented with fetal bovine serum (FBS) and L-glutamine.

[0396] Cell expansion: Upon thawing the vial, once the culture has reached a sufficient density for subculturing to the next cell expansion step, the cell culture is continuously expanded in growth medium (DMEM supplemented with FBS, L-glutamine, and hygromycin B) in a 5% CO2 incubator at 37°C. Cell expansion is continued until sufficient cells have grown to form one batch. One additional 10-layer CellStack (CS10) is prepared for each set of CS10, and the total viable cells are measured before initiating infection. Cell viability at the end of each subculturing should be 80% or higher. During the cell expansion step, cells are detached from the surface using TrypLE (recombinant trypsin). Furthermore, the culture is visually evaluated for cell morphology, cytopathic effects (CPE), and potential contamination.

[0397] Infection: Once the cell enlargement reaches the scale of one batch + one additional CS10, infect the cells by replacing the consumed medium with freshly prepared medium (DMEM supplemented with FBS and L-glutamine) containing FX201 and helper virus seeds. Before infection, randomly select one of the CS10s and measure the viable cell density and viability. Transfer the infected CS10s to a humidified CO2 incubator at 37°C and 5% CO2. The target volume for infection is approximately 600 g (or mL) per CS10. The total amount of FX201 and helper virus is for amplifying FX201 as needed.

[0398] Supply: Feed 116 infected cells in CS10 with fresh medium (DMEM supplemented with FBS and L-glutamine) and incubate further before harvesting. Visually inspect the culture for cell morphology, CPE, and potential contamination. Incubate for approximately 24 hours at 37°C in 5% CO2. The target volume for supply is approximately 600 g (or mL) per CS10.

[0399] Harvesting: At this stage, cells are expected to show signs of CPE and detach from the culture surface. To begin harvesting, gently detach the cells from CS10 by gently tapping to facilitate removal from the surface. Collect the detached cells, including the consumed medium, to obtain an untreated bulk harvest containing FX201. Samples are taken from the untreated bulk harvest for mycoplasma, bioburden, and in vitro exogenous virus testing. In-process testing of the untreated bulk harvest is performed to detect undetectable levels of mycoplasma (USP). <63> Ph.Eur.2.6.7, 1993 PTC) and exogenous viruses (in vitro assays for exogenous viruses using three cell lines) should be shown. Diffusion plate method, USP <61> Microbial counting tests using Ph.Eur.2.6.12 should detect less than 10 CFU / mL for TAMC and less than 10 CFU / mL for TYMC.

[0400] The untreated bulk harvest is clarified by centrifugation, and the supernatant containing FBS and residual hygromycin B in the culture medium is discarded. The cell pellet is then resuspended in a lysis buffer (100 mM Tris, 10% glycerol, pH 8.0). The resuspended cell pellet is frozen and held at -65°C or below, and then further purified.

[0401] Downstream processes Refining and manufacturing process A purification manufacturing process comprising cell lysis, benzoase digestion, clarification, ultracentrifugation, dialysis and formulation, and final filtration for the removal of impurities associated with the process and product is described herein. Appropriate controls are provided for each unit operation to help ensure consistency of the manufacturing process.

[0402] Lysis: FX201 is not lysogenic, and lysing of 116 infected cells is required to release viral particles for further processing. The resuspended cell pellet in lysis buffer (100 mM Tris, 10% glycerol, pH 8.0) is thawed by freezing and thawing by placing the container in a freezing bath (dry ice in isopropanol) and a warm water bath (37°C). At the completion of two freeze-thaw cycles and during the third freeze cycle, the resulting process intermediate can be kept below -65°C, and cells harvested from an additional set of CS10 can be pooled during the third thaw cycle to form one batch of formulation raw material.

[0403] Benzoase digestion: Cell lysates containing FX201 are treated with benzoase to digest any remaining host cell DNA. Benzoase is first diluted in a buffer containing 10 mM Tris and 10 mM MgCl2, and then added to the cell lysates for digestion. This process is carried out in a temperature-controlled water bath at 23°C, and the duration of benzoase digestion is recorded.

[0404] Clarification: The benzoase-digested cell lysates are clarified by centrifugation. After centrifugation, the supernatant is collected for further processing, and a sample is taken to measure the total number of virus particles and monitor the overall process yield.

[0405] Ultracentrifugation: The clarified cell lysates are purified by three rounds of cesium chloride (CsCl) ultracentrifugation to separate FX201 from impurities based on specific gravity. FX201 and helper viruses have different genome sizes (29.3kb and 36.0kb), resulting in different specific gravities and allowing for separation during ultracentrifugation. During each ultracentrifugation cycle, the clear separation of the band containing enriched FX201 from the impurity band is monitored for each tube.

[0406] For the first cycle of ultracentrifugation, the benzonase-digested and clarified cell lysates are diluted in dilution buffers (10 mM Tris and 10 mM MgCl2) and overlaid in a centrifugation tube containing two layers of CsCl at different specific densities. Two bands are present in the first cycle of ultracentrifugation, and enriched FX201 can be collected by extracting the lower band containing FX201 while discarding the upper band containing impurities. The collected bands containing FX201 are pooled, diluted, and purified by two further cycles of isodense ultracentrifugation with a single layer of CsCl.

[0407] Dialysis: Purified FX201 from the ultracentrifugation process is loaded into a dialysis cassette to further remove impurities, including CsCl and potential residual hygromycin B. After loading, the dialysis cassette is immersed in a container of formulation buffer (5% sucrose w / v, 0.5% ethanol v / v, 75 mM sodium chloride, 10 mM L-histidine, 10 mM Tris, 1.0 mM magnesium chloride, 0.02% polysorbate 80 v / v, 100 μM EDTA), replacing the entire contents of each container for each exchange. After four exchanges of the dialysis buffer, the purified FX201 is removed from the dialysis cassette, pooled, and diluted in the formulation buffer. In-process test samples are taken, and the concentration of total virus particles is measured by UV spectroscopy (OD260). The purified FX201 is frozen and held at -65°C or below before further processing.

[0408] Formulation and final filtration of the formulation raw material: To complete the formulation raw material manufacturing process, the frozen bottles of purified FX201 are thawed in a water bath. Upon thawing, the purified FX201 is thawed at the target concentration, for example, in a formulation buffer (5% sucrose w / v, 0.5% ethanol v / v, 75 mM sodium chloride, 10 mM L-histidine, 10 mM Tris, 1.0 mM magnesium chloride, 0.02% polysorbate 80 v / v, 100 μM EDTA) at a concentration of 2.3 × 10⁶ per 1 mL. 11Dilute to the virus particle (VP / mL). Filter the formulated FX201 through a 0.22 μm filter. Fill the resulting filtrate into polyethylene terephthalate copolymer, glycol modified (PETG) bottles with high-density polyethylene (HDPE) caps, and store frozen at -65°C or below to complete the production of the FX201 formulation raw material.

[0409] [Example 19] Development of a highly productive and reproducible manufacturing process for FX201, a novel helper-dependent adenovirus-based gene therapy for the treatment of osteoarthritis. Study Background: Gene therapy is a promising treatment option for osteoarthritis (OA) that shows potential to provide long-term efficacy and disease modification. FX201 (humantakinogene hadenovec) is a gene therapy under development for the treatment of OA delivered locally by intra-articular (IA) injection. FX201 is a novel helper-dependent adenovirus (HDAd), an engineered human serotype 5 adenovirus from which all viral genes have been removed. An expression cassette encoding the human interleukin-1 receptor antagonist (IL-1Ra) is inserted into the HDAd genome under the control of an inflammation-sensitive NF-κB promoter. Upon IA injection of FX201, cells in the joint are transduced to conditionally express the highly potent anti-inflammatory agent IL-1Ra, thereby reducing inflammation associated with OA of the knee.

[0410] Using a purposeful manufacturing process suitable for early development, four batches of the formulation were successfully produced, enabling pharmacokinetic, pharmacological, and Good Manufacturing Practice (GMP) clinical trials. Four batches of FX201, including one lot designed to express a rat ortholog of IL-1RA and three lots encoding human orthologs of IL-1RA, were produced in sufficient scale to enable Phase 1 clinical trials. Provided herein are manufacturing and product quality data across the four batches, demonstrating the reproducibility of the early-stage manufacturing process.

[0411] Methods: The manufacturing process described in Suzuki et al. (Hum Gene Ther. 2010;21(1), 120-126) was transferred to a contract manufacturing organization and adapted for GMP. Vials of cGMP cell bank containing the packaging cell line were thawed and continuously expanded using an adherent cell culture platform. Once the expanded cells reached a production-appropriate scale, FX201 and HV seed stocks were introduced into the culture. In the co-infected packaging cells, FX201 was grown in trans to borrow the HV genome and elements encoded in the packaging cells. The HV packaging signal was excised by recombinase, leaving most of the HV genome uncapsulated. Only the amplified genome with intact packaging signals was capsided. After incubation of the co-infection process, cells were harvested by centrifugation for downstream processing.

[0412] After harvesting, the supernatant is discarded by decantation, and the cells are subjected to a freeze-thaw cycle in a cell lysis buffer. The cell lysates are then digested with benzonase to allow for the removal of fragmented DNA by a subsequent centrifugation process. Process and product-related impurities, including rDNA, HCP, empty capsid, and residual HV, are removed from the benzonase-digested cell lysates by multiple ultracentrifugation cycles. The purified FX201 is then buffered by dialyzing into the final formulation buffer. For the final clinical material, the purified FX201 is diluted to the appropriate dose strength and aseptically packed into vials. Further details of the manufacturing process are also described in Example 18.

[0413] Three batches of non-clinical material and one batch of clinical-grade material were produced on two different scales. Two of the four batches, including one batch of HDAd vector encoding a rat variant of IL-1Ra, were used for toxicity and efficacy studies in rats. Each batch was analyzed for productivity, purification yield, and product quality, including physicochemical properties, infectivity and genomic titers, impurities associated with the product and process, transgene expression, and safety.

[0414] Results: Two nonclinical batches encoding human or rat variants of IL-1Ra, respectively, were produced at twice the scale of the following Operation (ENG) and GMP batches. The increased scale of the two nonclinical batches was due to the demand for materials needed to support animal testing, analytical development, implementation stability programs, and the establishment of reference standards.

[0415] Cell proliferation in adherent cultures: Packaging cells proliferated to the target density required for final expansion and subsequent co-infection. Target cell seeding density and culture time were controlled during the final expansion phase to enable consistent production across the four batches manufactured. Acceptable viable cell densities were achieved at the end of each passage (Figure 19A). Cell viability recovered from the initial thawing and maintained high viability throughout the expansion process (Figure 19B), showing sufficient cell proliferation for robust manufacturing operations. The total number of viable cells before infection was within 20% of the average for each of the four batches, which accounted for the differences in production scale.

[0416] Product yield in downstream processing steps: Batch productivity before purification, calculated using the total virus particle measurements at harvest across three batches of FX201 and one batch of rat ortholog, was within 30% on average, indicating similar starting volume (VP) for each purification lot. Post-purification batch yield was within 10% on average (Figure 20). This accounted for the differences in production scale. The higher overall yield observed in ENG runs was likely due to an underestimation of pre-purification batch productivity. Virus particles measured in the unpurified intermediate exhibit higher assay variability due to impurities that are later removed in the process.

[0417] Infectivity and Gene Expression: Human IL-1Ra expression was measured using a cell-based assay for three lots of FX201 and was within 20% of the mean based on ELISA endpoint measurements (Figure 21). While the infectivity of the ENG lot was lower than the other three lots, the ENG human IL-1Ra expression level was within 20% of the FX201 mean, suggesting that assay variability at this development stage likely caused the observed differences. (TCID) 50 ) and the ratio of genome copies to infectivity (referred to here as the ratio of viral particles to infectivity, VP / TCID) 50 The average of the ratio (also referred to as the ratio of virus particles to infectious units; these terms are used interchangeably) does not include data from ENG lots. TCID 50 The assay exhibits high assay variability, and the differences observed in ENG lots are within the range of assay variability. Assays based on different cells suggest consistent vector infection and transgene expression. IL-1Ra expression was detected in Tox (rat) (data not shown).

[0418] Analysis of impurities in the formulation raw materials: All process-related impurities other than rDNA (including host cell proteins (HCPs)) were mostly below detection or quantification levels and met clinically acceptable standards. The amount of residual DNA (rDNA) in the two nonclinical batches used in toxicity testing was approximately 150 times higher than that in the ENG and GMP batches. Two Tox lots with high rDNA levels (136 and 33 times higher than acceptable standards) were used in nonclinical studies to establish the efficacy, safety, and biodistribution of FX201 (Table 14). The established purification process sufficiently reduced residual HV and demonstrated the suitability of the purification process. Purified FX201 was characterized mostly as monomers in the final formulation.

[0419] [Table 14]

[0420] Conclusion: Productivity, purification yield, and product quality were generally consistent across three batches of FX201 and one batch of rat orthologs. These data indicate that the initial FX201 process is reproducible across multiple lots, demonstrating consistent productivity, purification yield, and product quality attributes. A single GMP formulation raw material batch yielded sufficient material to conduct a Phase I trial for patient safety and tolerability evaluation, encompassing three doses across a 100x total genome copy number range. Since one lot of formulation raw material is sufficient to supply the required formulations across the entire dose range of the Phase I trial, this demonstrates a viable manufacturing process for running the FX201 gene therapy program through clinical development.

Claims

1. A pharmaceutical composition comprising an adenovirus-based biological delivery and expression system formulated for intra-articular injection into human joints for the treatment of osteoarthritis or osteoarthritis conditions in human joints, or for the prevention of such conditions in humans identified as being at risk of developing osteoarthritis or osteoarthritis conditions, The adenovirus-based biological delivery and expression system includes a helper-dependent adenovirus vector genome copy (GC) containing a nucleic acid sequence encoding the human interleukin-1 receptor antagonist (IL-1Ra) protein, left and right reverse terminal repeats, an adenovirus packaging signal, and non-viral and non-coding stuffer nucleic acid sequences. The expression of nucleic acids encoding human IL-1Ra protein is regulated by an NF-κB-inducible promoter located upstream of the reading frame of the nucleic acid sequence encoding human IL-1Ra protein. The nucleic acid sequence of the adenovirus-based biological delivery and expression system, including the promoter, the nucleic acid sequence encoding IL-1Ra, the left and right reverse terminal repeats, the adenovirus packaging signal, and the non-viral non-coding stuffer nucleic acid sequence, is at least 95% homologous to the nucleic acid sequence of Sequence ID No.

7. The total dose of the pharmaceutical composition is 7 × 10 ９ ~2.8 x 10 １０ GC, 7x10 １０ ~2.8 x 10 １１ GC, or 7x10 １１ ~2.8 x 10 １２ A pharmaceutical composition containing a GC helper-dependent adenovirus vector.

2. The pharmaceutical composition according to claim 1, wherein the nucleic acid sequence of an adenovirus-based biological delivery and expression system, comprising a promoter, a nucleic acid sequence encoding IL-1Ra, left and right reverse terminal repeats, an adenovirus packaging signal, and a non-viral non-coding stuffer nucleic acid sequence, is at least 99% homologous to the nucleic acid sequence of Sequence ID No.

7.

3. The pharmaceutical composition according to claim 1, wherein the nucleic acid sequence of an adenovirus-based biological delivery and expression system, comprising a promoter, a nucleic acid sequence encoding IL-1Ra, left and right reverse terminal repeats, an adenovirus packaging signal, and a non-viral non-coding stuffer nucleic acid sequence, comprises the nucleic acid sequence of Sequence ID No.

7.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the nucleic acid encoding the IL-1Ra protein in the nucleic acid sequence of an adenovirus-based biological delivery and expression system comprises the nucleic acid of SEQ ID NO:

4.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the nucleic acid encoding the human IL-1Ra protein expresses a human IL-1Ra protein having an amino acid sequence that is at least 95% homologous to SEQ ID NO:

6.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein the nucleic acid encoding the human IL-1Ra protein expresses the human IL-1Ra protein containing the amino acid sequence of SEQ ID NO:

6.

7. Adenovirus-based biological delivery and expression systems are available for 1.4 × 10⁻⁶ years. １０ A pharmaceutical composition according to any one of claims 1 to 6, comprising GC.

8. Adenovirus-based biological delivery and expression systems are available for 1.4 × 10⁻⁶ years. １１ A pharmaceutical composition according to any one of claims 1 to 6, comprising GC.

9. A biological delivery and expression system based on adenovirus is 1.4×10 １２ The pharmaceutical composition according to any one of claims 1 to 6, comprising １２ GC.

10. A pharmaceutical composition according to any one of claims 1 to 9, wherein the volume of the composition is 1 mL to 5 mL.

11. A pharmaceutical composition according to any one of claims 1 to 9, wherein the volume of the composition is 5 mL.

12. a) Helper virus particles of less than 15%; b) Empty capsids with less than 10% empty content; c) Host cell proteins of 100 μg / ml or less; d) Host cell nucleic acids of 20 ng / ml or less; e) Endotoxins of 35 EU / ml or less; and f) 300GC / TCID ５０ The following ratios of virus particles to infection units A pharmaceutical composition according to any one of claims 1 to 11, further comprising:

13. A pharmaceutical composition according to any one of claims 1 to 12, used in a method comprising the following: a) A step of infecting human joint cells with a pharmaceutical composition comprising an adenovirus-based biological delivery and expression system; b) A step of expressing IL-1Ra within the joint.

14. The pharmaceutical composition according to claim 13, wherein the method comprises infecting articular cells once with an adenovirus-based biological delivery and expression system.

15. The pharmaceutical composition according to claim 13, wherein the method comprises infecting articular cells with an adenovirus-based biological delivery and expression system two or more times.

16. The pharmaceutical composition according to claim 15, wherein, when articular cells are infected with an adenovirus-based biological delivery and expression system two or more times, each infection comprises a helper-dependent adenovirus vector with a different number of genomic copies.

17. The pharmaceutical composition according to claim 15, wherein, when articular cells are infected with an adenovirus-based biological delivery and expression system two or more times, each infection comprises a helper-dependent adenovirus vector of the same number of genomic copies.

18. A pharmaceutical composition according to any one of claims 13 to 17, wherein infection of arthrocytes includes intra-articular injection.

19. The method is c) A step of treating or monitoring the progression of osteoarthritis or osteoarthritis in an infected joint after the expression of IL-1Ra protein in (b). A pharmaceutical composition according to any one of claims 13 to 18, further comprising:

20. The method is (d) If monitoring in (c) indicates that osteoarthritis or an osteoarthritis condition in the infected joint is not under control or treated, the step of continuing to administer the adenovirus-based biological delivery and expression system in that amount to the infected joint in (a); or If monitoring in (e)(c) indicates that osteoarthritis or the osteoarthritis condition has progressed in the infected joint, the process involves further adjusting the genomic copy number of the helper-dependent adenovirus vector in the adenovirus-based biological delivery and expression system and administering it to the osteoarthritis-affected joint in (a). The pharmaceutical composition according to claim 19, further comprising:

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the human joint is a human knee joint.

Images