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Novel gene therapy for rheumatoid arthritis with single local injection: adeno-associated virus-mediated delivery of A20/TNFAIP3
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Qin Zhang1, 2, Fang-Xing Yu3, Yang-Lin Wu1, 2, Cheng-Yuan Yang2, Nai-Cheng Liu2, Xu Zhu2, Pi-Ming Zhao3, Zhong-Ya Wang3, Jun Lin1, 2, *
Military Medical Research | 2023, 10(3) : 418 - 420
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Military Medical Research | 2023, 10(3): 418-420
• LETTER TO THE EDITOR •
Novel gene therapy for rheumatoid arthritis with single local injection: adeno-associated virus-mediated delivery of A20/TNFAIP3
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Qin Zhang1, 2, Fang-Xing Yu3, Yang-Lin Wu1, 2, Cheng-Yuan Yang2, Nai-Cheng Liu2, Xu Zhu2, Pi-Ming Zhao3, Zhong-Ya Wang3, Jun Lin1, 2, *
Affiliations
  • 1Department of Orthopaedics, Suzhou Dushu Lake Hospital, Dushu Lake Hospital Affiliated to Soochow University, Medical Center of Soochow University, Suzhou 215001, Jiangsu, China
  • 2Department of Orthopaedics, the First Affiliated Hospital of Soochow University, Soochow University, Suzhou 215006, Jiangsu, China
  • 3Department of Gene Therapy, CureGenetics Co., Ltd., Suzhou 215021, Jiangsu, China
Published: 2023-06-10 doi: 10.1186/s40779-022-00393-0
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Adeno-associated virus  /  Gene therapy  /  Rheumatoid arthritis  /  TNF-α-induced protein 3 (A20)
Qin Zhang, Fang-Xing Yu, Yang-Lin Wu, Cheng-Yuan Yang, Nai-Cheng Liu, Xu Zhu, Pi-Ming Zhao, Zhong-Ya Wang, Jun Lin. Novel gene therapy for rheumatoid arthritis with single local injection: adeno-associated virus-mediated delivery of A20/TNFAIP3[J]. Military Medical Research, 2023 , 10 (3) : 418 -420 . DOI: 10.1186/s40779-022-00393-0
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Dear Editor,
Multiple experiments have established TNF-α-induced protein 3 (A20/TNFAIP3) as a critical regulator associated with rheumatoid arthritis (RA)[1,2]. The lack of TNF-α-induced protein 3 (A20) promotes the NOD-like receptor protein 3 (NLRP3) inflammasome and induces spontaneous arthritis, while increase of A20 reduces the secretion of IL-1β and favors immunological tolerance[3,4]. Hence, we investigate the feasibility of recombinant adeno-associated virus 6 (rAAV6)-mediated A20 gene therapy in a collagen-induced arthritis (CIA) model.
We first investigated cytomegalovirus (CMV) promoter-regulated gene delivery (Additional file 1: Fig. S1). rAAV6 (Additional file 1: Fig. S1a, b) was transfected into 293 T cells (Additional file 1: Fig. S2a) to enhance A20 expression (Additional file 1: Fig. S2b). A 10 μl of volume rAAV6-CMV-A20 containing 1×1012 viral genomes (vg) was injected into the left knee, ankle, and tarsal area of CIA mice, while the same dose of rAAV6-CMV-EGFP was injected into the right side (Additional file 1: Fig. S2c). rAAV6 was widely distributed in various cell types (Additional file 1: Fig. S2d) and significantly enhanced A20 expression until 5 weeks after injection (Fig. 1a). Notably, rAAV6-CMV-A20 decreased the clinical arthritis score, paw thickness and total porosity (P<0.001), increased the bone volume-to-tissue volume ratio (BV/TV, P<0.001), trabecular number (Tb.N, P=0.008), and trabecular thickness (Tb.Th, P=0.002) (Fig. 1b–d, Additional file 1: Fig. S2e–j), and suppressed pannus formation, bone erosion, and cartilage destruction (Fig. 1e). We also found that rAAV6-CMV-A20 significantly suppressed the expression of NLRP3, caspase-1, and IL-1β (Additional file 1: Fig. S2k–m). Thus, the results verified our hypothesis that rAAV6-mediated A20 overexpression is an effective RA therapy.
The CMV promoter may attract safety concerns. Therefore, considering that macrophages and fibroblast-like synoviocytes account for the primary components of synovial tissues, especially in RA, we tested which cell type predominantly overexpressed A20. We constructed rAAV6-COL1α-A20 (Additional file 1: Fig. S1c) targeting fibroblast-like synoviocytes and rAAV6-SP146-A20 (Additional file 1: Fig. S1e) targeting macrophages[5]. A 10 μl volume of rAAV6-COL1α-A20 or rAAV6-SP146-A20 (1×1012 vg) was injected into the left knee, ankle, and tarsal area, while the same dose of rAAV6-COL1α-EGFP (Additional file 1: Fig. S1d) or rAAV6-SP146-EGFP (Additional file 1: Fig. S1f) was injected into the same areas on the right side (Additional file 1: Fig. S3a). In contrast to rAAV6-COL1α-EGFP, rAAV6-COL1α-A20 exhibited nearly no effect on suppressing RA symptoms. However, rAAV6-SP146-A20 showed anti-rheumatic therapeutic effects compared with rAAV6-SP146-EGFP. Similar to rAAV6-CMV-A20, rAAV6-SP146-A20 reduced the clinical arthritis score, paw thickness (Fig. 1f, g; Additional file 1: Fig. S3b, c), total porosity (Additional file 1: Fig. S3d), bone erosion activity (Additional file 1: Fig. S3e–g), pannus formation, and cartilage destruction (Fig. 1h, i). rAAV6-SP146-EGFP was well transfected into RAW264.7 cells (Fig. 1j) and exhibited dominant distribution in macrophages (Additional file 1: Fig. S3h). rAAV6-SP146-A20 significantly enhanced A20 expression (Fig. 1k, l) and the collective results further indicated the therapeutic benefits of rAAV6-mediated A20 overexpression. Importantly, macrophages were found to be responsible for the rAAV6-mediated A20 overexpression.
The anti-rheumatic benefits of rAAV6-SP146-A20 were further verified by simultaneous injection of rAAV. CIA mice were injected with rAAV6-SP146-EGFP or rAAV6-SP146-A20 on both sides (1×1012 vg, Additional file 1: Fig. S3i). As expected, rAAV6-SP146-A20 significantly relieved the arthritis symptoms (Additional file 1: Figs. S3j–o, S4a–c). Furthermore, we tested whether the therapeutic effect was dependent on viral genomes. CIA mice were injected with rAAV6-SP146-EGFP at a dose of 1×1012 vg (EGFP) or rAAV6-SP146-A20 at a dose of 1×108 vg (E8), 1×1010 vg (E10), or 1×1012 vg (E12, Additional file 1: Fig. S3p). The results revealed that the protective effect of rAAV6-SP146-A20 was dose-dependent. The E12 group exhibited the lowest clinical arthritis score and increases in paw thickness, bone erosion activity, pannus formation, and cartilage destruction (Additional file 1: Figs. S3q–v, S4d–e). As the treatment dose increased, so did the expression of A20, while NLRP3, caspase-1, and IL-1β were gradually inhibited (Additional file 1: Fig. S4f–g).
Our hypothesis that A20 overexpression is a reasonable strategy for the treatment of RA was initially evidenced. We not only demonstrated the therapeutic effects against RA of ubiquitous CMV promoter-driven delivery of A20, but more importantly, we identified the key role of macrophage-like synovial cells in responding to rAAV6-mediated gene delivery of A20.
Abbreviations
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A20/TNFAIP3: TNF-α-induced protein 3; BV/TV: Bone volume-to-tissue volume ratio; CMV: Cytomegalovirus; CIA: Collagen-induced arthritis; NLRP3: NOD-like receptor protein 3; RA: Rheumatoid arthritis; Tb.N: Trabecular number; Tb.Th: Trabecular thickness.
Supplementary information
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The online version contains supplementary material available at https://doi.org/10.1186/s40779-022-00393-0.
Additional file 1. Materials and methods. Fig. S1: Construction of rAAV expression plasmids. Fig. S2: Therapeutic effect of rAAV-CMV-A20. Fig. S3: Verification of the therapeutic effect of rAAV6-SP146-A20. Fig. S4: rAAV6-SP146-A20 inhibits NLRP3-mediated inflammation in CIA mice.
Acknowledgements
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Not applicable.
Author contributions
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QZ performed the experiments, analyzed the data, and wrote the manuscript. FXY established rAAV6. YLW, CYY, NCL, and XZ assisted with the cell experiments and data analysis. PMZ and ZYW designed rAAV6 and supervised the development of rAAV6. JL conceived and designed the study. All authors read and approved the final version of the manuscript.
Availability of data and materials
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All data needed to evaluate the conclusions in the paper are present in the paper or the Additional files. The data are available from the corresponding author on reasonable request.
Declarations
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Ethics approval and consent to participate
Not applicable.
Consent for publication
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Not applicable.
Competing interests
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Fang-Xing Yu, Pi-Ming Zhao, and Zhong-Ya Wang (CureGenetics Co., Ltd.) are responsible for the encapsulation and identification of the rAAV required for the experiment. The authors declare that they have no competing interest for this work.
Author details
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1Department of Orthopaedics, Suzhou Dushu Lake Hospital, Dushu Lake Hospital Affiliated to Soochow University, Medical Center of Soochow University, Suzhou 215001, Jiangsu, China. 2Department of Orthopaedics, the First Affiliated Hospital of Soochow University, Soochow University, Suzhou 215006, Jiangsu, China. 3Department of Gene Therapy, CureGenetics Co., Ltd., Suzhou 215021, Jiangsu, China.
Funding
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  • National Natural Science Foundation of China(81871789)
  • National Natural Science Foundation of China(82172387)
  • Natural Science Foundation of Jiangsu Province(BK20180052)
  • Gusu Health Talents Program(GSWS2020023)
References
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1.
Vande Walle L, van Opdenbosch N, Jacques P, Fossoul A, Verheugen E, Vogel P, et al. Negative regulation of the NLRP3 inflammasome by A20 protects against arthritis. Nature. 2014;512(7512):69-73.
2.
Matmati M, Jacques P, Maelfait J, Verheugen E, Kool M, Sze M, et al. A20 (TNFAIP3) deficiency in myeloid cells triggers erosive polyarthritis resembling rheumatoid arthritis. Nat Genet. 2011;43(9):908-12.
3.
Polykratis A, Martens A, Eren RO, Shirasaki Y, Yamagishi M, Yamaguchi Y, et al. A20 prevents inflammasome-dependent arthritis by inhibiting macrophage necroptosis through its ZnF7 ubiquitin-binding domain. Nat Cell Biol. 2019;21(6):731-42.
4.
Duong BH, Onizawa M, Oses-Prieto JA, Advincula R, Burlingame A, Malynn BA, et al. A20 restricts ubiquitination of pro-interleukin-1β protein complexes and suppresses NLRP3 inflammasome activity. Immunity. 2015;42(1):55-67.
5.
Kang WS, Kwon JS, Kim HB, Jeong HY, Kang HJ, Jeong MH, et al. A macrophage-specific synthetic promoter for therapeutic application of adiponectin. Gene Ther. 2014;21(4):353-62.
Appendix
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Year 2023 volume 10 Issue 3
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Article Info
doi: 10.1186/s40779-022-00393-0
  • Online Date：2025-11-27
  • Published：2023-06-10
Article Data
Affiliations
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Funding
National Natural Science Foundation of China(81871789)
National Natural Science Foundation of China(82172387)
Natural Science Foundation of Jiangsu Province(BK20180052)
Gusu Health Talents Program(GSWS2020023)
Affiliations
    1Department of Orthopaedics, Suzhou Dushu Lake Hospital, Dushu Lake Hospital Affiliated to Soochow University, Medical Center of Soochow University, Suzhou 215001, Jiangsu, China
    2Department of Orthopaedics, the First Affiliated Hospital of Soochow University, Soochow University, Suzhou 215006, Jiangsu, China
    3Department of Gene Therapy, CureGenetics Co., Ltd., Suzhou 215021, Jiangsu, China

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表12种不同金属材料的力学参数
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Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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