Selection of a novel AAV2/TNFAIP3 vector for local suppression of islet xenograft inflammation

被引：5
作者：
Zammit, Nathan W. [1 ,2 ]
Seeberger, Karen L. [3 ]
Zamerli, Jad [1 ]
Walters, Stacey N. [1 ]
Lisowski, Leszek [4 ,5 ]
Korbutt, Gregory S. [3 ]
Grey, Shane T. [1 ,2 ]
机构：
[1] Garvan Inst Med Res, Immunol Dept, Darlinghurst, NSW, Australia
[2] Univ New South Wales Sydney, St Vincents Clin Sch, Fac Med, Sydney, NSW, Australia
[3] Univ Alberta, Dept Surg, Edmonton, AB, Canada
[4] Univ Sydney, Childrens Med Res Inst, Translat Vectorol Unit, Westmead, NSW, Australia
[5] Mil Inst Med, Lab Mol Oncol & Innovat Therapies, Warsaw, Poland
来源：
XENOTRANSPLANTATION | 2021年 / 28卷 / 03期
基金：
英国医学研究理事会; 澳大利亚国家健康与医学研究理事会;
关键词：
A20; AAV2; adeno‐ associated virus; adenovirus; beta cell replacement; cytoprotective; differentiation; inflammation; islet; maturation; neonatal porcine islets; NF‐ κ B; NPI; porcine; TNFAIP3; transplant; type; 1; diabetes; NEONATAL PORCINE ISLETS; FACTOR-KAPPA-B; ADENOASSOCIATED-VIRUS; GENE DELIVERY; ENDOTHELIAL-CELLS; TRANSPLANTATION; A20; XENOTRANSPLANTATION; THERAPY; TRANSDUCTION;
D O I：
10.1111/xen.12669
中图分类号：
R-3 [医学研究方法]; R3 [基础医学];
学科分类号：
1001 ;
摘要：
Background Neonatal porcine islets (NPIs) can restore glucose control in mice, pigs, and non-human primates, representing a potential abundant alternative islet supply for clinical beta cell replacement therapy. However, NPIs are vulnerable to inflammatory insults that could be overcome with genetic modifications. Here, we demonstrate in a series of proof-of-concept experiments the potential of the cytoplasmic ubiquitin-editing protein A20, encoded by the TNFAIP3 gene, as an NPI cytoprotective gene. Methods We forced A20 expression in NPI grafts using a recombinant adenovirus 5 (Ad5) vector and looked for impact on TNF-stimulated NF-kappa B activation and NPI graft function. As adeno-associated vectors (AAV) are clinically preferred vectors but exhibit poor transduction efficacy in NPIs, we next screened a series of AAV serotypes under different transduction protocols for their ability achieve high transduction efficiency and suppress NPI inflammation without impacting NPI maturation. Results Forcing the expression of A20 in NPI with Ad5 vector blocked NF-kappa B activation by inhibiting I kappa B alpha phosphorylation and degradation, and reduced the induction of pro-inflammatory genes Cxcl10 and Icam1. A20-expressing NPIs also exhibited superior functional capacity when transplanted into diabetic immunodeficient recipient mice, evidenced by a more rapid return to euglycemia and improved GTT compared to unmodified NPI grafts. We found AAV2 combined with a 14-day culture period maximized NPI transduction efficiency (>70% transduction rate), and suppressed NF-kappa B-dependent gene expression without adverse impact upon NPI maturation. Conclusion We report a new protocol that allows for high-efficiency genetic modification of NPIs, which can be utilized to introduce candidate genes without the need for germline engineering. This approach would be suitable for preclinical and clinical testing of beneficial molecules. We also report for the first time that A20 is cytoprotective for NPI, such that A20 gene therapy could aid the clinical development of NPIs for beta cell replacement.
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页数：12
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