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

Abstract

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‐κB 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‐κB activation by inhibiting IκBα 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‐κB‐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.

Heterogenous expression of endocrine and progenitor cells within the neonatal porcine pancreatic lobes–Implications for neonatal porcine islet xenotransplantation

“…Intact porcine pancreases (n = 23 female and n = 25 male) were excised from anesthetized 1 to 3 days old Duroc-Landrace neonatal piglets from the University of Alberta Swine Research Centre. 1,6,[33][34][35] During excision, the CL and bridge were dissected from the intestine, the SL was detached from the spleen and the DL was dissected from the duodenum by severing the duct (Figure 1A and B). To examine gross morphology, the whole pancreas (n = 4 female and n = 4 male) and dissected lobes were weighed, then assessed for total cellular insulin content as previously described.…”

“…Immunostaining and histological assessment was performed on 5 μm paraffin sections from each lobe as previously described. 1,6,[33][34][35] Primary antibodies included; insulin (IR00261-2; Agilent, Santa Clara CA, USA), proliferating cell nuclear antigen (PCNA; M0879; Agilent), cytokeratin 7 (CK-7; M701801-2; Agilent), somatostatin (SOM) (A0566; Agilent), pancreatic polypeptide (PPP) (A0619; Agilent), glucagon (G2654; MilliporeSigma, Oakville ON, Canada), pancreatic duodenal homeobox 1 (PDX1; ab47267; AbCAM, Cambridge, MA USA), PCNA For immunofluorescence, AlexaFluor conjugates were cover slipped with ProlongGold Anti-Fade with 4' ,6-diamidino-2-phenylindole (DAPI;ThermoFisher). Positive controls were new born pig pancreas sections and negative controls were sections incubated with secondary antibodies alone without primary antibody staining.…”

“…NPIs were isolated from lobes (n = 24 either sex), as previously described. 1,6,[33][34][35] Following an initial culture in Ham's F-10 Nutrient Mixture (MilleporeSigma) for 3 days, islets were differentiated utilizing a multi-step protocol until 15 days post-isolation. 1,6,33 At Day 3 and 15, total cellular DNA and insulin contents were assessed.…”

“…1,6,[33][34][35] Following an initial culture in Ham's F-10 Nutrient Mixture (MilleporeSigma) for 3 days, islets were differentiated utilizing a multi-step protocol until 15 days post-isolation. 1,6,33 At Day 3 and 15, total cellular DNA and insulin contents were assessed. 1,6 Cellular DNA content was determined in duplicate utilizing the CyQuant Cell Proliferation Assay (ThermoFisher) following the manufacturer's instructions.…”

Updateon xenotransplantation for May/June 2021

“…Functional studies on the A20 enzyme have shown that it is a major regulator of the inflammatory cascade that occurs by inhibiting NF‐κB activation and forced expression on mouse or human islets does not impair insulin secretion 52,53 . To validate the use of A20 in porcine NICCs, Grey's group developed a series of recombinant adenoviral vectors that expressed A20 and they transduced these into NICCs and measured their suppressive abilities and transduction efficiency 54 . Twenty‐four hours post forced expression of A20 on NICCs resulted in reduced IκBα degradation and phosphorylation as well as the suppression of islet inflammatory mRNAs such as Cxcl10, Icam1, Il1b, and Il6.…”