Gene drug for treating arthritis

Abstract

A gene drug for treating arthritis. Disclosed are a neuropeptide Y (NPY)-based gene therapy, and a composition using rAAV as a vector for the treatment of gouty arthritis or rheumatoid arthritis, and a use thereof.

Description

A gene drug for treating arthritis

[0001] priority

[0002] This application claims the benefit of and priority to Chinese application No. 2023109895054, filed on August 7, 2023, the entire contents of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present disclosure belongs to the field of biomedicine, and in particular relates to the manufacture and application of a composition for treating arthritis using rAAV as a vector for NPY-based gene therapy. Background Art

[0004] Rheumatoid arthritis (RA) is an autoimmune disease with synovitis as its pathological basis and may eventually lead to joint deformities. Among the clinical manifestations of patients, joint lesions are the main ones. The initial main symptoms are morning stiffness, swelling and pain in the joints. As the disease progresses, joint deformities may develop, affecting the patient's normal joint function. The treatment of rheumatoid arthritis mainly depends on the specific situation of the patient. Conventional treatment includes drug therapy and surgical treatment. Rheumatoid arthritis treatment often uses immunosuppressants to delay the progression of the disease, such as disease-modifying antirheumatic drugs (DMARDs), nonsteroidal antiinflammatory drugs (NSAIDs), glucocorticosteroids (GCs), biological DMARDs (bDMARDs), etc. Commonly used biological agents include infliximab, etanercept and adalimumab. AbbVie's Humira and Su Lixin of Innovent Biologics Both are adalimumab injections, which are indicated for rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, juvenile idiopathic arthritis, etc., but are not suitable for gouty arthritis.

[0005] Although both are arthritis diseases, gouty arthritis and rheumatoid arthritis have significant differences in causes, symptoms, prevalent sites, and prevalent populations, and there are also differences in treatment methods.

[0006] Gout arthritis is a common and complex type of arthritis caused by the accumulation of monosodium urate (MSU) crystals in joints and periarticular tissues. It is a non-autoimmune disease. Gout is caused by hyperuricemia, which is a result of purine metabolism disorders and an imbalance in uric acid metabolism. Gout arthritis is caused by the accumulation of MSU crystals, which triggers activation of the innate immune system, leading to a strong inflammatory response in the local joints and periarticular tissues, manifested primarily by redness, swelling, heat, and pain. Gout arthritis can be divided into primary and secondary gouty arthritis based on its etiology. The cause of primary gouty arthritis is still unclear, but it may be related to genetic factors and is often associated with conditions such as hypertension, coronary artery disease, and obesity. Secondary gouty arthritis is often caused by the patient's underlying medical conditions, such as kidney disease and bone hyperplasia. Gout arthritis can be divided into acute and chronic gouty arthritis based on the severity of onset. Patients with acute gouty arthritis often experience sudden onset of joint pain at night. The onset is rapid, with pain, edema, redness, swelling, and inflammation in the joints. The pain gradually subsides and disappears, lasting from a few days to a few weeks. Chronic gouty arthritis typically develops from acute gouty arthritis, with symptoms gradually worsening, leading to limited joint function and deformities, severely impacting quality of life. It is estimated that the prevalence of gouty arthritis in my country is approximately 1.14%, with approximately 16 million people suffering from gout nationwide, with middle-aged men aged 40 and above being the most susceptible. With rising living standards and changing lifestyles, the incidence of gout is increasing, with a younger prevalence, posing a serious health risk to patients. Common treatments for gouty arthritis include nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. However, these drugs only provide pain relief and often cause numerous adverse side effects and a high relapse rate. These drugs are rapidly eliminated through metabolism, necessitating frequent oral or intravenous administration to maintain therapeutic effects. Long-term treatment imposes a significant financial burden on patients, and repeated injections increase pain and the likelihood of adverse reactions. How to reduce the frequency of medication and reduce adverse reactions is the main bottleneck in the current treatment of gouty arthritis.

[0007] Although gouty arthritis and rheumatoid arthritis rarely coexist, there are some case reports in the literature (JEBAKUMAR A, UDAYAKUMAR PD, CROWSON CS, et al. Occurrence of gout in rheumatoid arthritis: it does happen! A population-based study[J]. Int J Clin Rheumatol, 2013, 8: 433-437; Lao Danhua. Analysis of 5 cases of rheumatoid arthritis and gout[J]. Clinical Meta-Collection, 2011, 26(14): 1236-1238; OLARU L, SOONG L, DHILLON S, et al. Coexistent rheumatoid arthritis and gout: a case series and review of the literature[J]. Clin Rheumatol, 2017, 36(12): 2835-2838; MARTINEZ-CORDERO E, BESSUDO-BABANI A, TREVINO PEREZ SC, et al. Concomitant gout and rheumatoid arthritis [J]. J Rheumatol, 1988, 15(8): 1307-1311). Studies have shown that although the incidence rate is lower than that of the normal population, patients with rheumatoid arthritis may have gout (OLARU L, SOONG L, DHILLON S, et al. Coexistent rheumatoid arthritis and gout: a case series and review of the literature[J]. Clin Rheumatol, 2017, 36(12): 2835-2838; MARTINEZ-CORDERO E, BESSUDO-BABANI A, TREVINO PEREZ SC, et al. Concomitant gout and rheumatoid arthritis[J]. J Rheumatol, 1988, 15(8): 1307-1311). How to treat cases of the two diseases is also a problem currently facing arthritis treatment.

[0008] Summary of the Invention

[0009] The present disclosure provides use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY) in the preparation of a medicament for treating gouty arthritis in a subject.

[0010] The present disclosure provides the use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY) in the preparation of a medicament for treating gouty arthritis and rheumatoid arthritis in a subject.

[0011] The present disclosure provides uses of a nucleic acid encoding neuropeptide Y (NPY), a vector or vector system comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY), or a composition containing the same in the preparation of a medicament for treating gouty arthritis in a subject.

[0012] The present disclosure provides uses of a nucleic acid encoding neuropeptide Y (NPY), a vector or vector system comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY), or a composition containing the same in the preparation of a medicament for treating gouty arthritis and rheumatoid arthritis in a subject.

[0013] In another aspect, the present disclosure provides a method for treating gouty arthritis in a subject, administering to the subject a therapeutically effective amount of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY).

[0014] In another aspect, the present disclosure provides a method for treating gouty arthritis and rheumatoid arthritis in a subject, administering to the subject a therapeutically effective amount of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY).

[0015] In another aspect, the present disclosure provides use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY) in a method of treating gouty arthritis in a subject.

[0016] In another aspect, the present disclosure provides use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY) in a method for treating gouty arthritis and rheumatoid arthritis in a subject.

[0017] In some embodiments, the neuropeptide Y described in the present disclosure is mammalian NPY or a functional variant thereof, preferably rat NPY (rNPY), human NPY (hNPY), mature human NPY (hmNPY), mouse NPY (musNPY), or a functional variant thereof.

[0018] In some embodiments, the hNPY comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO: 1.

[0019] In some embodiments, the nucleic acid encoding the hNPY comprises the nucleic acid sequence shown in SEQ ID NO: 2, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO: 2.

[0020] In some embodiments, the hmNPY comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO: 3.

[0021] In some embodiments, the nucleic acid encoding the hmNPY comprises the nucleic acid sequence shown in SEQ ID NO: 4, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO: 4.

[0022] In some embodiments, the rNPY comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO:5.

[0023] In some embodiments, the nucleic acid encoding the rNPY comprises the nucleic acid sequence shown in SEQ ID NO: 6, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO: 6.

[0024] In some embodiments, the musNPY comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO:7.

[0025] In some embodiments, the nucleic acid encoding the musNPY comprises a nucleic acid sequence as shown in SEQ ID NO:8, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO:8.

[0026] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0027] In some embodiments, the composition further comprises a second drug that can treat gouty arthritis.

[0028] In some embodiments, the virus is an adeno-associated virus.

[0029] In some embodiments, the adeno-associated virus comprises a capsid, and the serotype of the capsid is selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10 or AAVrh10, preferably AAV5 or AAV9.

[0030] In some embodiments, the vector is an adeno-associated viral vector comprising:

[0031] 1) Promoter;

[0032] 2) nucleic acid encoding neuropeptide Y (NPY);

[0033] 3) Two ITR sequences, preferably the ITR sequences of AAV2 virus.

[0034] In some embodiments, the subject has gouty arthritis or rheumatoid arthritis; in some embodiments, the subject has both gouty arthritis and rheumatoid arthritis.

[0035] In some embodiments, the drug is administered to the patient by subcutaneous injection, intramuscular injection, intra-articular injection, or ganglion injection; preferably, subcutaneous injection, intramuscular injection, ganglion injection, or intra-articular injection.

[0036] In some embodiments, the injection method is intra-articular injection, and the intra-articular injection is intra-articular injection of the knee or the ankle.

[0037] In some embodiments, the injection site of the knee joint cavity is in the middle of the patellar ligament at the level of the inferior edge of the patella.

[0038] In some embodiments, the location of the ankle joint injection is the lateral ankle joint cavity. Beneficial effects

[0039] Compared with long-term, multiple oral or injections of nonsteroidal anti-inflammatory drugs and corticosteroids, gene therapy using NPY is a completely new treatment method that can address the limitations of existing treatments for gouty arthritis, prolong the half-life of the drug in the body, reduce the number of doses, reduce the occurrence of drug resistance, reduce the occurrence of adverse reactions, and alleviate patients' pain and economic burden. It has the potential to become a promising, attractive, and cost-effective alternative. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present disclosure can be more fully understood with reference to the following drawings.

[0041] FIG1 shows a schematic diagram of the structure of the ssAAV-XMAN01 vector.

[0042] FIG2 shows a schematic diagram of the structure of the ssAAV-XMAN02 vector.

[0043] FIG3 shows a schematic diagram of the structure of the ssAAV-NC vector.

[0044] FIG4 shows the transcription levels of XMAN01 and XMAN02 in cells.

[0045] FIG5 shows a schematic diagram and statistical graphs showing the inhibitory effects of ganglion injection of XMAN01, XMAN02 and XMAN03 on joint swelling in mice with MSU-induced gouty arthritis.

[0046] FIG6 shows a schematic diagram and statistical graphs showing the inhibitory effect of intraganglionic injection of XMAN01 on collagen-induced joint swelling in rheumatoid arthritis mice.

[0047] FIG7 shows a schematic diagram and statistical graph showing the inhibitory effect of ankle joint intra-articular injection of XMAN01 and XMAN02 on joint swelling in MSU-induced gouty arthritis mice.

[0048] FIG8 shows a schematic diagram and statistical graph showing the inhibitory effects of ankle joint cavity injection of XMAN01 and subcutaneous injection of HUMIRA on joint swelling in MSU-induced gouty arthritis mice.

[0049] FIG9 shows HE staining images of ankle joint sections of MSU-induced gouty arthritis mice injected with XMAN01 and HUMIRA.

[0050] FIG10 shows the effects of injection of XMAN01 and HUMIRA on synovial hyperplasia and inflammatory cell infiltration in the joint cavity of mice with MSU-induced gouty arthritis.

[0051] FIG11 shows the effects of injection of XMAN01, XMAN02 and HUMIRA on pro-inflammatory and anti-inflammatory factors in the joints of mice with MSU-induced gouty arthritis.

[0052] FIG12 shows the expression of target genes in mice after ankle joint intra-articular injection of XMAN01.

[0053] FIG13 shows a schematic diagram and statistical graph showing the inhibitory effects of ankle joint cavity injection of XMAN01 and intraperitoneal injection of HUMIRA on collagen-induced joint swelling in rheumatoid arthritis mice.

[0054] FIG14 shows a schematic diagram and statistical graph showing the effects of ankle joint cavity injection of XMAN01 and intraperitoneal injection of HUMIRA on arthritis symptoms in the upper and lower limbs of collagen-induced rheumatoid arthritis mice.

[0055] FIG15 shows HE staining images of ankle joint sections of collagen-induced rheumatoid arthritis mice injected with XMAN01 and intraperitoneally injected with HUMIRA.

[0056] FIG16 shows the effects of injection of XMAN01 and HUMIRA on synovial hyperplasia and inflammatory cell infiltration in the joint cavity of rheumatoid arthritis mice induced by collagen. DETAILED DESCRIPTION

[0057] The following description of the present disclosure is intended only to illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and it should be understood that these equivalent embodiments are intended to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0058] The present disclosure provides use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY) in the preparation of a medicament for treating gouty arthritis in a subject.

[0059] The present disclosure provides the use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY) in the preparation of a medicament for treating rheumatoid arthritis in a subject.

[0060] The present disclosure provides a use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY) in the preparation of a medicament for treating gouty arthritis and rheumatoid arthritis in a subject suffering from both gouty arthritis and rheumatoid arthritis.

[0061] The present disclosure provides uses of a nucleic acid encoding neuropeptide Y (NPY), a vector or vector system comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY), or a composition containing the same in the preparation of a medicament for treating gouty arthritis in a subject.

[0062] The present disclosure provides uses of a nucleic acid encoding neuropeptide Y (NPY), a vector or vector system comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY), or a composition containing the same in the preparation of a medicament for treating rheumatoid arthritis in a subject.

[0063] The present disclosure provides uses of a nucleic acid encoding neuropeptide Y (NPY), a vector or vector system comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY), or a composition containing the same in the preparation of a medicament for treating gouty arthritis and rheumatoid arthritis in a subject suffering from both gouty arthritis and rheumatoid arthritis.

[0064] In another aspect, the present disclosure provides a method for treating gouty arthritis in a subject, administering to the subject a therapeutically effective amount of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY).

[0065] In another aspect, the present disclosure provides a method for treating rheumatoid arthritis in a subject by administering to the subject a therapeutically effective amount of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY).

[0066] In another aspect, the present disclosure provides a method for treating gouty arthritis and rheumatoid arthritis in a subject, wherein a therapeutically effective amount of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY) is administered to the subject, wherein the subject suffers from both gouty arthritis and rheumatoid arthritis.

[0067] In another aspect, the present disclosure provides use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY) in a method of treating gouty arthritis in a subject.

[0068] In another aspect, the present disclosure provides use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY) in a method of treating rheumatoid arthritis in a subject.

[0069] In another aspect, the present disclosure provides use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY) in a method for treating gouty arthritis and rheumatoid arthritis in a subject who suffers from both gouty arthritis and rheumatoid arthritis.

[0070] In some embodiments, the construct contains a nucleic acid encoding neuropeptide Y (NPY). The construct can generally be constructed by inserting the nucleic acid encoding neuropeptide Y (NPY) into a suitable expression vector, and a person skilled in the art can select a suitable expression vector.

[0071] In some embodiments, the cell comprising the nucleic acid encoding neuropeptide Y (NPY) can be a host cell, the host cell comprising the above-mentioned construct or the above-mentioned nucleic acid integrated into the genome, or the host cell comprising a virus as described in any of the above items, such as an adeno-associated virus. Representative examples of suitable host cells include mammalian cells (such as CHO or COS), plant cells, human cells (human embryonic kidney cells such as HEK293FT), bacterial cells (such as Escherichia coli, Streptomyces, Salmonella typhimurium), fungal cells (such as yeast), insect cells (such as Sf9), etc. A person skilled in the art can select a suitable host based on the teachings herein. Preferably, the host cell is an animal cell, and more preferably a human cell. The host cell can be a cultured cell or a primary cell, that is, directly isolated from an organism (such as a human). The host cell can be an adherent cell or a suspended cell, that is, a cell grown in suspension. Furthermore, the adeno-associated virus further includes a heterologous nucleic acid sequence encoding a target product, and the heterologous nucleic acid sequence encoding the target product can be encapsulated and carried by various capsid proteins. The above-mentioned heterologous nucleic acid sequence encoding the product of interest can generally be a construct, and the construct can generally contain a nucleic acid encoding the product of interest. The construct can generally be constructed by inserting the nucleic acid encoding the product of interest into a suitable expression vector. Those skilled in the art can select a suitable expression vector. For example, the above-mentioned expression vector can include but is not limited to pAAV-CAG, pAAV-TRE, pAAV-EF1a, pAAV-GFAP promoter, pAAV-Lgr5 promoter, pAAV-Sox2 promoter expression vectors, etc. In the present disclosure, when the adeno-associated virus encodes the heterologous nucleic acid sequence of the product of interest, the adeno-associated virus contains a capsid, and the viral vector carries a transgene encoding a gene product, and the transgene is regulated by a regulatory sequence that directs its expression in the host cell.

[0072] In some embodiments, the present disclosure further provides an engineered host cell transformed with the adeno-associated virus described above. The engineered host cell comprises the adeno-associated virus described above. The host cell can be a eukaryotic cell and / or a prokaryotic cell.

[0073] In some embodiments, the cell containing the nucleic acid encoding neuropeptide Y (NPY) can also be a therapeutic cell, which is introduced into a subject to express neuropeptide Y (NPY) in the subject to achieve a therapeutic effect.

[0074] In some embodiments, the virus encoding the nucleic acid of neuropeptide Y (NPY) can be an adeno-associated virus. The adeno-associated virus comprises a capsid, and the serotype of the capsid is selected from AAV2, AAV5 or AAV9. In some embodiments, the serotype of the AAV is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10 or AAVrh10. In some embodiments, the AAV vector comprises one or more AAV inverted terminal repeat (ITR) sequences flanking it. In some embodiments, the heterologous nucleic acid is flanked by two AAV ITRs. In some embodiments, the AAV ITR is an ITR of the serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10. In some embodiments, the AAV ITR is an AAV2 ITR. In some embodiments, the ITRs and capsid of the AAV particle are derived from the same AAV serotype. In some embodiments, the ITRs and capsid are derived from AAV2. In other embodiments, the ITRs and capsid of the AAV virus particle are derived from different AAV serotypes. The term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term "AAV virus" or "AAV virus particle" or "AAV vector particle" refers to a virus particle of an AAV vector that contains at least one AAV capsid protein and one encapsidated polynucleotide.

[0075] In some embodiments, the AAV vector comprises one or more promoters, and / or enhancers, and / or polyadenylation signals. In some embodiments, the vector is an adeno-associated viral vector comprising:

[0076] 1) Promoter;

[0077] 2) nucleic acid encoding neuropeptide Y (NPY);

[0078] 3) Two ITR sequences, preferably the ITR sequences of AAV2 virus.

[0079] In some embodiments, the vector system of the present disclosure can be an adeno-associated virus vector system, which comprises a packaging plasmid, and the packaging plasmid further comprises a rep gene segment of an adeno-associated virus. The rep gene segment comprises an intron, and the intron comprises a transcription termination sequence. The packaging plasmid, expression plasmid, and helper virus plasmid are transferred into a host cell, and the nucleic acid sequences therein are all integrated into the host cell to produce the adeno-associated virus. In some embodiments, all of the nucleic acid sequences are integrated together at a single locus in the cell genome of the host cell. In some embodiments, the nucleic acid sequences encoding various genes exist as separate expression cassettes; the nucleic acid sequences encoding the rep and cap genes exist in the same or different expression cassettes.

[0080] Neuropeptide Y (NPY) belongs to the NPY family of neuroendocrine peptides, which also includes peptide YY (PYY) and pancreatic polypeptide. NPY is widely present throughout the body. In the central nervous system, it is distributed in the hippocampus, cerebral cortex, hypothalamus, thalamus, brainstem, and cerebellum. NPY is also widely present in peripheral tissues such as the retina, smooth muscle, intestine, bone marrow, and thymus.

[0081] In some embodiments, the neuropeptide Y described in the present disclosure is mammalian NPY or a functional variant thereof, preferably rat NPY (rNPY), human NPY (hNPY), mature human NPY (hmNPY), mouse NPY (musNPY), or a functional variant thereof.

[0082] Specifically, a functional variant of NPY refers to a polypeptide fragment obtained by substituting, deleting, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the amino acid sequence of mammalian NPY, or by adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the N-terminus and / or C-terminus, but still having the function of NPY. The functional variant may have a sequence identity of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with NPY, such as any of the sequences in SEQ ID NOs: 1-8.

[0083] In some embodiments, the hNPY comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO: 1.

[0084] In some embodiments, the nucleic acid encoding the hNPY comprises the nucleic acid sequence shown in SEQ ID NO: 2, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO: 2.

[0085] In some embodiments, the hmNPY comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO: 3.

[0086] In some embodiments, the nucleic acid encoding the hmNPY comprises the nucleic acid sequence shown in SEQ ID NO: 4, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO: 4.

[0087] In some embodiments, the rNPY comprises the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO:5.

[0088] In some embodiments, the nucleic acid encoding the rNPY comprises the nucleic acid sequence shown in SEQ ID NO: 6, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO: 6.

[0089] In some embodiments, the musNPY comprises the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with SEQ ID NO:7.

[0090] In some embodiments, the nucleic acid encoding the musNPY comprises a nucleic acid sequence as shown in SEQ ID NO: 10, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to SEQ ID NO: 8.

[0091] In some embodiments, the composition containing neuropeptide Y (NPY) further includes a pharmaceutically acceptable carrier or excipient. The excipient may include various excipients and diluents. These excipients themselves are not essential active ingredients and are not overly toxic after administration. Suitable excipients should be well known to those skilled in the art. The acceptable carriers include, for example, sterile water or physiological saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (phosphoric acid, citric acid, other organic acids, etc.), preservatives, surfactants (such as PEG, Tween), chelating agents (such as EDTA), adhesives, etc. In addition, it may also contain other low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine, and lysine; sugars or carbohydrates such as polysaccharides and monosaccharides; and sugar alcohols such as mannitol or sorbitol. When preparing aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, appropriate solubilizers such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), non-ionic surfactants (Tween 80, HCO-50), etc. can be used in combination.

[0092] In some embodiments, the composition further comprises a second drug that can treat gouty arthritis, and the second drug is packaged in the same or different medium or kit as neuropeptide Y (NPY). In some embodiments, the second drug can be administered before, after, or simultaneously with the administration of neuropeptide Y (NPY), a nucleic acid encoding neuropeptide Y (NPY), a vector comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY).

[0093] The term "subject" generally includes humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., which can benefit from treatment using the formulation, kit or combination formulation. In some embodiments, the subject suffers from both gouty arthritis and rheumatoid arthritis. In some embodiments, the subject is a mammal, preferably a human.

[0094] In some embodiments, the adeno-associated virus or pharmaceutical composition provided by the present disclosure can be adapted to a suitable mode of administration and administered subcutaneously, intramuscularly, in the joint system, in the nervous system, or in the circulatory system, such as in blood or body fluids. For example, the composition is suitable for intraocular, intravenous, intraarterial, subcutaneous, intratracheal or inhalation administration. Those skilled in the art can select a suitable dose according to the mode of administration. In some embodiments, the drug is administered by subcutaneous injection, intramuscular injection, ganglion injection, or intraarticular injection.

[0095] The dosage of the active ingredient of the adeno-associated virus or pharmaceutical composition generally depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the virus or pharmaceutical composition as the active ingredient can generally be 1-1000 mg / kg / day, 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or greater than 500 mg / kg / day.

[0096] The terms "include", "comprising", etc. should be understood as having an inclusive meaning rather than an exclusive or exhaustive meaning; that is, the meaning is "including but not limited to".

[0097] The term "therapeutically effective amount" generally refers to an amount that can achieve the effect of treating the diseases listed above after an appropriate administration period.

[0098] The term "treatment" should be understood in its broadest sense. The term "therapeutic" does not necessarily imply that a mammal is treated until full recovery. Similarly, "prophylaxis" does not necessarily mean that a subject will not ultimately contract a disease condition. Thus, treatment and prevention include alleviating the symptoms of a particular condition or preventing or reducing the risk of a particular condition developing. The term "prevention" can be understood to mean reducing the severity of an episode of a particular condition. Treatment can also include reducing the severity of an existing condition or the frequency of acute episodes.

[0099] In the present disclosure, the subject or individual for therapeutic or prophylactic treatment is preferably a mammal, such as, but not limited to, a human, a primate, livestock (e.g., sheep, cattle, horses, donkeys, pigs), a pet (e.g., a dog, a cat), a laboratory animal (e.g., a mouse, rabbit, rat, guinea pig, hamster), or a captive wild animal (e.g., a fox, a deer). The subject is preferably a primate. The subject is most preferably a human.

[0100] Before further describing the specific embodiments of the present disclosure, it should be understood that the scope of protection of the present disclosure is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present disclosure are for the purpose of describing specific specific embodiments rather than for the purpose of limiting the scope of protection of the present disclosure; in the present disclosure and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0101] When numerical ranges are given in the examples, it should be understood that, unless otherwise specified in the present disclosure, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meanings as those commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the examples of the present disclosure may also be used to implement the present disclosure, based on the prior art knowledge of those skilled in the art and the disclosure.

[0102] Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields, which are well described in the existing literature.

[0103] Statistical analysis

[0104] GraphPad Prism 8.0 software was used for data processing and statistical analysis. The statistical level was set at 5% or p ≤ 0.05, and the mean and standard error (mean ± SEM) of each analysis indicator were calculated. A p ≤ 0.05 was considered statistically significant.

[0105] In order to enable those skilled in the art to better understand the solutions of the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all of the embodiments.

[0106] Example 1: Construction of plasmid vector

[0107] Construction of AAV vector plasmids containing human NPY (hNPY), mature human NPY (hmNPY), and rat NPY (rNPY) genes:

[0108] Based on the amino acid sequence of hNPY (P01303) published on UniProt (SEQ ID NO:1), its nucleic acid sequence (SEQ ID NO:2) was designed using human codon preference. A BamHI restriction site was introduced at the 5' end, and an EcoRV restriction site was introduced at the 3' end. The gene was named XMAN01. The gene was synthesized by Beijing Qingke Biotechnology Co., Ltd. After digesting XMAN01 and the ssAAV-CAG plasmid with BamHI and EcoRV, respectively, the ssAAV-XMAN01 vector was constructed using conventional molecular biology techniques, including T4 DNA ligase ligation, transformation, and single-clone screening. A schematic diagram of its structure is shown in Figure 1. Positive clones were verified by PCR and sequencing, and high-quality plasmid DNA was obtained using an endotoxin-free plasmid extraction kit (MACHEREY-NAGEL GmbH & Co. KG, Cat. No. 740420.50) for future use.

[0109] Based on the amino acid sequence of hNPY published on UniProt (SEQ ID NO:1) and the amino acid sequence of hmNPY reported by Ana Santos-Carvalho et al. (SEQ ID NO:3), a nucleic acid sequence (SEQ ID NO:4) was designed according to human codon usage. A BamHI restriction site was added to the 5' end of the primer, and an EcoRV restriction site was introduced at the 3' end. The gene was named XMAN02. The gene was synthesized by Beijing Qingke Biotechnology Co., Ltd. After digesting XMAN02 and the ssAAV-CAG plasmid with BamHI and EcoRV, the ssAAV-XMAN02 vector was constructed using standard molecular biology techniques, including T4 DNA ligase ligation, transformation, and single-clone screening. A schematic diagram of its structure is shown in Figure 2. Positive clones were verified by PCR and sequencing, and high-quality plasmid DNA was obtained using an endotoxin-free plasmid extraction kit (MACHEREY-NAGEL GmbH & Co. KG) for future use.

[0110] The ssAAV-XMAN03 vector was constructed based on the amino acid (SEQ ID NO: 5) and nucleic acid (SEQ ID NO: 6) sequences of rNPY (P07808) published on UniProt.

[0111] Based on the amino acid sequence of eGFP (A0A076FL24) published in the UniProt database (SEQ ID NO:9), a nucleic acid sequence (SEQ ID NO:10) was designed based on human codon preference. A BamH I restriction site was added to the 5' end of the primer, and an EcoR V restriction site was introduced at the 3' end. The resulting sequence was designated NC. The gene was synthesized by Beijing Qingke Biotechnology Co., Ltd. After digesting the NC and ssAAV-CAG plasmids with BamH I and EcoR V, the ssAAV-NC vector was constructed using conventional molecular biology techniques, including T4 DNA ligase ligation, transformation, and single-clone screening. A schematic diagram of its structure is shown in Figure 3. Positive clones were verified by PCR and sequencing, and high-quality plasmid DNA was obtained using an endotoxin-free plasmid extraction kit (MACHEREY-NAGEL GmbH & Co. KG) for future use.

[0112] Table 1. Sequence information

[0113] Example 2: Preparation and identification of recombinant AAV virus

[0114] Recombinant AAV virus was prepared using a three-plasmid packaging system. The specific method is as follows: helper plasmid (pHelper), AAV Rep (type 2) and Cap (type 9) protein expression plasmid (AAV2 / 9) (Tong, D. et al. Single-dose AAV-based vaccine induces a high level of neutralizing antibodies against SARS-CoV-2 in rhesus macaques. Protein & Cell 14, 69–73, doi: 10.1093 / procel / pwac020 (2022)) and ITR expression plasmid (XMAN01, XMAN02, XMAN03, NC) were used to obtain high-quality plasmid DNA using an endotoxin-free plasmid extraction kit (MACHEREY-NAGEL GmbH & Co. KG). HEK293T cells were subcultured in 10 cm culture dishes. When the confluence reached approximately 90%, the cells were replaced with 4 mL of reduced serum medium (DMEM medium + 4% FBS + 1% Pen Strep, where DMEM medium was purchased from Gibco, FBS was purchased from Natocor, Pen Strep and PS were purchased from Gibco) and cultured for 2 hours. Transfection reagent was prepared by adding 1 mL of transfection reagent (1 mL Opti-MEM medium + 40 μg transfection agent + 10 μg pHelper + 5 μg AAV2 / 9 + 5 μg XMAN01 / XMAN02 / XMAN03 / NC, where Opti-MEM medium was purchased from Gibco) to each dish. The transfection agent used was polyethyleneimine (PEI, purchased from Polysciences). Approximately 6 hours after transfection, the culture dish was replaced with 10 mL of reduced serum medium and the cells were returned to the incubator for continued culture. The virus in the supernatant was collected twice: the first collection was after 3 days of culture. After collecting the cell culture supernatant, fresh low-serum culture medium was added, and the cells were returned to the incubator for continued culture. The cell supernatant was centrifuged at 3000 rpm for 10 minutes to remove cell debris; the second collection was after 7 days of culture. The cell culture supernatant was collected and centrifuged at 3000 rpm for 10 minutes to remove cell debris.

[0115] Recombinant AAV virus was prepared using a three-plasmid packaging system as follows: a helper plasmid (pHelper) (Tong, D. et al. Single-dose AAV-based vaccine induces high levels of neutralizing antibodies against SARS-CoV-2 in rhesus macaques. Protein & Cell 14, 69–73, doi:10.1093 / procel / pwac020(2022)), an AAV Rep (type 2) and Cap (type 5) protein expression plasmid (AAV2 / 5) (Miaoling Biotechnology), and an ITR expression plasmid (XMAN01, XMAN02) were used to obtain high-quality plasmid DNA for later use using an endotoxin-free plasmid extraction kit (MACHEREY-NAGEL GmbH & Co. KG). HEK293T cells were subcultured in 10 cm culture dishes and, when confluence reached approximately 90%, were replaced with 4 mL of low-serum medium (DMEM + 4% FBS + 1% PS) and cultured for 2 hours. Transfection reagent preparation: Add 1 mL of transfection reagent (1 mL Opti-MEM + 40 μg transfection agent + 10 μg pHelper + 5 μg AAV2 / 5 + 5 μg XMAN01 / XMAN02) to each dish. Use polyethyleneimine (PEI) as the transfection agent. Approximately 6 hours after transfection, replace the dish with 10 mL of reduced-serum medium and return the dish to the incubator. Harvest the virus from the supernatant twice: the first harvest occurs after 3 days of culture. After harvesting the cell culture supernatant, fresh reduced-serum medium is added. Return the dish to the incubator for further culture, and the supernatant is centrifuged at 3000 rpm for 10 minutes to remove cell debris. The second harvest occurs after 7 days of culture. The cell culture supernatant is harvested and centrifuged at 3000 rpm for 10 minutes to remove cell debris.

[0116] Purification of recombinant AAV virus: First, the cell supernatant was concentrated to approximately 10 mL using a centrifugal filter device (Millipore, Cat No. 910024). Then, pure recombinant AAV was obtained by iodixanol gradient centrifugation. After elution with PBS, the purified AAV was aliquoted and stored in a -80°C refrigerator until use.

[0117] Titer determination of recombinant AAV virus: The purified recombinant AAV virus is first tested for viral titer by qPCR, and then the morphology, titer, and purity of the recombinant AAV virus are further verified by transmission electron microscopy. In this way, recombinant AAV virus with acceptable titer and purity is obtained.

[0118] Example 3: Determination of the transcriptional expression of XMAN01 and XMAN02 in cells

[0119] ssAAV-XMAN01 and ssAAV-XMAN02 plasmids were transfected into HEK293T cells respectively, and the cells were collected and the expression of mRNA was detected by RT-qPCR. The specific operation is as follows: HEK293T cells were subcultured in a 10 cm culture dish. When the confluence reached 70% to 90%, 4 mL of low serum culture medium (DMEM culture medium + 4% FBS + 1% PS) was changed to culture for 2 hours. Transfection reagent preparation: 1 mL of transfection reagent (1 mL Opti-MEM + 40 μg transfection agent + 20 μg ssAAV-XMAN01 / ssAAV-XMAN02) was added to each dish. Polyethyleneimine (PEI) was used as the transfection agent. About 6 hours after transfection, the liquid in the culture dish was replaced with 10 mL of low serum culture medium, and continued to be placed back in a 37°C, 5% CO2 incubator for culture, and untransfected cells were set as a control group. After 72 hours of culture, the cells were collected and RNAeasy TM After RNA was extracted using a viral RNA extraction kit (Biyuntian, R0035L), the RNA was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme). Real-time PCR amplification reaction was then performed using the cDNA as a template to relatively quantify the mRNA level in the cells.

[0120] The results are shown in Figure 4. Compared with the control group, the transcription levels of XMAN01 and XMAN02 were significantly increased, and the transcription level of XMAN01 was higher than that of XMAN02.

[0121] Example 4: Efficacy of XMAN01, XMAN02, and XMAN03 in an animal model of gouty arthritis

[0122] Sixty SPF male C57BL / 6J mice, 7-8 weeks old (purchased from Beijing Weitonglihua Experimental Animal Breeding Co., Ltd.), weighing 22-25 g, were examined before injection. Qualified animals were injected. The specific groups of experimental animals are shown in Table 2 below.

[0123] Table 2. Experimental animal groups

[0124] Since it takes a certain amount of time for the expression of the target gene to reach a stable level after AAV injection, AAV drug injection was performed on the first day, and gouty arthritis models were established in the successfully injected animals on the 22nd day.

[0125] The specific method for modeling gouty arthritis in C57BL / 6J mice is as follows: the mice were injected with sodium urate (MSU, Sigma-Aldrich, Cat No. U2875, 0.5 mg MSU crystals suspended in 20 ul endotoxin-free PBS) at a dose of 0.5 mg / mouse into the ankle joint for modeling, and the control group was injected with an equal volume of PBS into the ankle joint (see Yin C, Liu B, Wang P, et al. Eucalyptol alleviates inflammation and pain responses in a mouse model of gout arthritis[J]. Br J Pharmacol, 2020, 177(9): 2042-2057).

[0126] The ankle joints of the mice were observed, photographed, and measured 1 hour before modeling and 48 hours after modeling. The average value was calculated after three consecutive measurements. The joint measurement data were statistically analyzed.

[0127] The results of ganglion injection are shown in Figure 5. Compared with the control group, the ankle joint thickness of the model group increased significantly, and the degree of joint swelling was obvious; compared with the model group, the injection of XMAN01 and XMAN03 significantly inhibited the increase in ankle joint thickness and successfully improved the degree of joint swelling.

[0128] Example 5: Efficacy of XMAN01 in an animal model of rheumatoid arthritis

[0129] Twenty-four SPF male DBA / 1 mice, 6-7 weeks old (purchased from Beijing Weitonglihua Experimental Animal Breeding Co., Ltd.), weighing 19-23 g, were examined before injection. Qualified animals were injected. The specific groups of experimental animals are shown in Table 3 below.

[0130] Table 3. Experimental animal groups

[0131] Since it takes a certain amount of time for the expression of the target gene to reach a stable level after AAV injection, AAV drug injection was performed on the first day, and rheumatoid arthritis models were established in the successfully injected animals on the 22nd day.

[0132] The specific method for establishing the rheumatoid arthritis model in DBA / 1 mice is as follows: the mice were given the first immunization 3 weeks after administration, with a subcutaneous injection of 100 μL of a mixed collagen solution (Chondrex, 20012) and Freund's complete adjuvant (Chondrex, 7001) into the tail; and the mice were given the second immunization 3 weeks after the first immunization, with a subcutaneous injection of 100 μL of a mixed collagen solution and Freund's incomplete adjuvant (Chondrex, 7002) into the tail (see Brand, D., Latham, K. & Rosloniec, E. Collagen-induced arthritis. Nat Protoc 2, 1269–1275 (2007)).

[0133] Ten days after the secondary immunization, the ankle joints of the mice were observed and measured weekly. Three measurements were taken and the average was calculated. Statistical analysis of the joint measurement data was performed. The results of ganglion injection are shown in Figure 6. Compared with the control group, the ankle joint thickness of the modeling group increased significantly, and redness and swelling of the toes, soles, and joints were more pronounced. Compared with the modeling group, injection of XMAN01 significantly inhibited the increase in joint thickness and improved redness and swelling of the toes, soles, and joints.

[0134] Example 6: Efficacy of XMAN01 and XMAN02 in a gouty arthritis animal model using a novel administration route

[0135] Sixty SPF male C57BL / 6J mice, 7-8 weeks old (purchased from Beijing Weitonglihua Experimental Animal Breeding Co., Ltd.), weighing 22-25 g, were examined before injection. Qualified animals were injected. The specific groups of experimental animals are shown in Table 4 below.

[0136] Table 4. Experimental animal groups

[0137] Because it takes time for the expression of the target gene to reach a stable level after AAV injection, AAV drug injection was performed on day 1, and gouty arthritis models were established in successfully injected animals on day 22. The HUMIRA group was injected 1 hour before gouty arthritis modeling on day 22.

[0138] The specific method for modeling gouty arthritis in C57BL / 6J mice is as follows: the mice were injected with sodium urate (MSU, Sigma-Aldrich, Cat No. U2875, 0.5 mg MSU crystals suspended in 20 ul endotoxin-free PBS) at a dose of 0.5 mg / mouse into the ankle joint for modeling, and the control group was injected with an equal volume of PBS into the ankle joint (see Yin C, Liu B, Wang P, et al. Eucalyptol alleviates inflammation and pain responses in a mouse model of gout arthritis[J]. Br J Pharmacol, 2020, 177(9): 2042-2057).

[0139] Morphology: The ankle joints of mice were observed, photographed, and measured 1 hour before modeling and 48 hours after modeling. The average value was calculated after three consecutive measurements. Statistical analysis of joint measurement data was performed.

[0140] The results of ankle joint cavity injection are shown in Figures 7 and 8. Compared with the control group, the ankle joint thickness of the model group increased significantly, and the degree of joint swelling was obvious. Compared with the model group, the injection of XMAN01 significantly inhibited the increase in joint thickness and successfully improved the degree of joint swelling. The injection of HUMIRA had no significant inhibitory effect.

[0141] Pathology: After euthanasia, the ankle joints of mice were removed and fixed in 4% paraformaldehyde (biosharp) at 4°C for 48 h. After washing four times with PBS (biosharp), the joints were immersed in EDTA decalcification solution for decalcification (Seville). The decalcification solution was changed every 2-3 days. After decalcification, the joints were dehydrated in 30% sucrose (Shengong) until they sank to the bottom. 15 μm thick frozen sections were then sliced ​​and stained with HE. After staining, the sections were photographed for histopathological changes, synovial hyperplasia was scored, and inflammatory cells were counted and analyzed using Image J.

[0142] The results are shown in Figures 9 and 10. Compared with the control group, the synovium in the ankle joint cavity of the modeling group proliferated significantly (Figures 9A and 10A), and there was a large amount of inflammatory cell infiltration (Figures 9B and 10B); compared with the modeling group, injection of XMAN01 significantly inhibited synovial hyperplasia and inflammatory cell infiltration, while injection of HUMIRA had no significant inhibitory effect.

[0143] Inflammatory factors: After cardiac perfusion, the ankle joints of mice were removed, the skin was removed, and 600 μL of ice-cold lysis buffer (from RNAeasy TM Viral RNA extraction kit, Biyuntian, R0035L), frozen and ground in a fully automatic sample rapid grinder, and then RNAeasyTM RNA was extracted using a viral RNA extraction kit. The extracted RNA was quantified and then reverse-transcribed into cDNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme). The cDNA, double-distilled water, and SYBR Mix (Vazyme) were mixed into an amplification system for qPCR determination of IL-6 and IL-10 mRNA expression changes. Three replicate wells were used for each assay.

[0144] The results are shown in Figure 11. Compared with the control group, the expression level of the pro-inflammatory cytokine IL-6 in the modeling group was significantly increased; compared with the modeling group, the injection of XMAN01 and XMAN02 significantly reduced the expression of the pro-inflammatory cytokine IL-6, and significantly increased the expression of the anti-inflammatory cytokine IL-10; the injection of HUMIRA failed to significantly increase the expression of the anti-inflammatory factor IL-10.

[0145] Based on the results of morphology, pathology and inflammatory factors, injection of XMAN01 has a better inhibitory effect than HUMIRA on ankle joint swelling, joint cavity synovial hyperplasia and inflammation in the MSU-induced gouty arthritis mouse model.

[0146] Example 7: Expression of target gene in mice

[0147] After the mouse heart was perfused in Example 6, the ankle joint was removed, the skin was removed, and 600 μL of ice-cold lysis buffer (from RNAeasy TM Viral RNA extraction kit, Biyuntian, R0035L), frozen and ground in a fully automatic sample rapid grinder, and then RNAeasy TM RNA was extracted using a viral RNA extraction kit. The extracted RNA was quantified and then reverse-transcribed into cDNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme). The cDNA, double-distilled water, and SYBR Mix (Vazyme) were mixed into an amplification system for qPCR determination of NPY mRNA expression changes. Three replicate wells were used for each assay.

[0148] The results are shown in FIG12 . Compared with the control group and the model group, higher expression of the target gene was detected in the XMAN01 group.

[0149] Example 8: Efficacy of XMAN01 and XMAN02 in a rheumatoid arthritis animal model using a novel administration route

[0150] Forty-eight SPF male DBA / 1 mice, 6-7 weeks old (purchased from Beijing Weitonglihua Experimental Animal Breeding Co., Ltd.), weighing 22-25 g, were examined before injection. Qualified animals were injected. The specific groups of experimental animals are shown in Table 5 below.

[0151] Table 5. Grouping of experimental animals

[0152] The specific method for establishing the rheumatoid arthritis model in DBA / 1 mice is as follows: the mice were initially immunized on day 1 by subcutaneous injection of 100 μL of a mixed collagen solution (Chondrex, 20012) and Freund's complete adjuvant (Chondrex, 7001) into the tail; three weeks after the initial immunization, the mice were secondary immunized by subcutaneous injection of 100 μL of a mixed collagen solution and Freund's incomplete adjuvant (Chondrex, 7002) into the tail (see Brand, D., Latham, K. & Rosloniec, E. Collagen-induced arthritis. Nat Protoc 2, 1269–1275 (2007)).

[0153] Mice were injected with AAV drugs on day 1 (the day of initial immunization), and the HUMIRA group was injected with drugs once a week after the onset of symptoms until the mice were euthanized.

[0154] Ten days after the second immunization, the morphological indicators of the mice were observed and measured weekly. The specific morphological indicators observed are as follows:

[0155] Ankle joint thickness: The ankle joints of mice were observed, photographed, and measured. The average value of three measurements was taken, and the joint measurement data were statistically analyzed.

[0156] Arthritic Index (AI): Scoring was performed regularly according to the literature, using a scale of 0 to 4 based on the degree of redness, swelling, and joint mobility of the mouse's limbs. Each limb was scored separately, with a maximum score of 4. No redness or swelling: 0; redness or swelling of one toe joint without involving adjacent toe joints: 1; swelling of two or more toe joints or the plantar: 2; swelling of three or more toe joints or the ankle and below: 3; swelling of all paws, local ulceration, or loss of joint mobility: 4. The sum of the scores for all four limbs constituted the AI ​​(see Brand, D., Latham, K. & Rosloniec, E. Collagen-induced arthritis. Nat Protoc 2, 1269–1275 (2007)).

[0157] The measurement and scoring results of ankle joint injection are shown in Figures 13 and 14. Compared with the control group, the modeling group showed a significant increase in ankle joint thickness, with significant redness and swelling in the toes, soles, and joints, and also in the upper limbs. Compared with the modeling group, injection of XMAN01 and HUMIRA significantly inhibited the increase in joint thickness and improved redness and swelling in the toes, soles, and joints of the upper and lower limbs.

[0158] Pathology: After euthanasia, the ankle joints of mice were removed and fixed in 4% paraformaldehyde (biosharp) at 4°C for 48 h. The joints were washed four times with PBS (biosharp) and then immersed in EDTA decalcification solution for decalcification (Seville). The decalcification solution was changed every 2-3 days. After decalcification, the joints were placed in 30% sucrose (Shengong) for dehydration until they sank to the bottom. 15 μm thick frozen sections were then cut and stained with HE. After staining, the sections were photographed to observe the histopathological changes, score the synovial hyperplasia, and count the inflammatory cells in the ankle joint sections using Image J and analyze the differences.

[0159] The results are shown in Figures 15 and 16. Compared with the control group, the synovium in the ankle joint cavity of the modeling group proliferated massively, destroying the cartilage (Figures 15A and 16A), and there was a large amount of inflammatory cell infiltration (Figures 15B and 16B); compared with the modeling group, the injection of XMAN01 significantly inhibited the synovial hyperplasia and inflammatory cell infiltration, while the injection of HUMIRA significantly inhibited the inflammatory cell infiltration, but had no obvious inhibitory effect on synovial hyperplasia.

[0160] Based on the comprehensive morphological and pathological results, the injection of XMAN01 is as effective as HUMIRA in inhibiting joint redness and swelling, and inflammatory cell infiltration in the joint cavity of the collagen-induced rheumatoid arthritis mouse model, and is better than HUMIRA in inhibiting synovial hyperplasia in the joint cavity.

[0161] Incorporated by Reference

[0162] Each patent and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0163] Equivalence

[0164] The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above-described embodiments should be considered in all cases as illustrative rather than limiting of the invention described herein. The scope of the present disclosure is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalents of the claims are intended to be embraced therein.

Claims

1. Use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY) in the preparation of a medicament for treating gouty arthritis in a subject.

2. Use of neuropeptide Y (NPY) or a composition containing neuropeptide Y (NPY) in the preparation of a medicament for treating gouty arthritis and rheumatoid arthritis in a subject.

3. Use of a nucleic acid encoding neuropeptide Y (NPY), a vector or vector system comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY), or a composition comprising the above substances in the preparation of a medicament for treating gouty arthritis in a subject.

4. Use of a nucleic acid encoding neuropeptide Y (NPY), a vector or vector system comprising a nucleic acid encoding neuropeptide Y (NPY), a virus comprising a nucleic acid encoding neuropeptide Y (NPY), or a cell comprising a nucleic acid encoding neuropeptide Y (NPY), or a composition comprising the above substances in the preparation of a medicament for treating gouty arthritis and rheumatoid arthritis in a subject.

5. The use according to any one of claims 1 to 4, wherein the neuropeptide Y is NPY from mammals or a functional variant thereof, preferably rNPY, hNPY, hmNPY, musNPY, or a functional variant thereof.

6. The use according to claim 5, wherein the hNPY comprises the amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity with SEQ ID NO:

1.

7. The use according to claim 5, wherein the nucleic acid encoding the hNPY comprises the nucleic acid sequence as shown in SEQ ID NO: 2, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity with SEQ ID NO:

2.

8. The use according to claim 5, wherein the hmNPY comprises the amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity with SEQ ID NO:

3.

9. The use according to claim 5, wherein the nucleic acid encoding the hmNPY comprises the nucleic acid sequence as shown in SEQ ID NO:4, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity with SEQ ID NO:

4.

10. The use according to claim 5, wherein the rNPY comprises the amino acid sequence as shown in SEQ ID NO:5, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity with SEQ ID NO:

5.

11. The use according to claim 5, wherein the nucleic acid encoding the rNPY comprises the nucleic acid sequence as shown in SEQ ID NO:6, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity with SEQ ID NO:

6.

12. The use according to claim 5, wherein the musNPY comprises the amino acid sequence as shown in SEQ ID NO:7, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity with SEQ ID NO:

7.

13. The use according to claim 5, wherein the nucleic acid encoding the musNPY comprises the nucleic acid sequence as shown in SEQ ID NO:8, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% sequence identity with SEQ ID NO:

8.

14. The use according to any one of claims 1 to 4, wherein the composition further comprises a pharmaceutically acceptable carrier.

15. The use according to any one of claims 1 to 4, wherein the composition further comprises a second drug for treating gouty arthritis.

16. The use according to claim 3 or 4, wherein the virus is an adeno-associated virus.

17. The use according to claim 16, wherein the adeno-associated virus comprises a capsid, and the serotype of the capsid is selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10 or AAVrh10, preferably AAV5 or AAV9.

18. The use according to claim 3 or 4, wherein the vector is an adeno-associated virus vector, comprising: 1) Promoter; 2) Nucleic acid encoding neuropeptide Y (NPY); 3) Two ITR sequences, preferably the ITR sequences of AAV2 virus.

19. The use of claim 1 or 3, wherein the subject suffers from gouty arthritis or rheumatoid arthritis.

20. The use according to claim 2 or 4, wherein the subject suffers from both gouty arthritis and rheumatoid arthritis.

21. The use according to any one of claims 1 to 4, wherein the drug is administered subcutaneously, intramuscularly, in the joint system, in the nervous system, or in the circulatory system; preferably subcutaneously, intramuscularly, in ganglia, or intraarticularly.

22. The use according to claim 21, wherein the intra-articular injection is a knee intra-articular injection or an ankle intra-articular injection.

23. The use according to claim 22, wherein the injection site of the knee joint cavity is in the middle of the patellar ligament at the level of the lower edge of the patella.

24. The use according to claim 22, wherein the location of the ankle joint cavity injection is the lateral ankle joint cavity.

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