Thu September 21 from 17:15 to 18:15
Room: Foyer, Exhibit Hall

Posters: Poster Networking Session

#Poster #7 Anti-apoptotic Effects of HO1 and A20 Expression in Endothelial Cells of Multi-transgenic Pigs for Xenotransplantation
Presenter
Dandro, Amy S.
Senior Research Associate, Revivicor, Inc — Blacksburg, United States
Abstract
IXA 2017 abstracts have been published in Xenotransplantation journal

(2017), IXA 2017- Abstracts of the 14th Congress of the International Xenotransplantation Association, Baltimore, USA. Xenotransplantation, 24: n/a, e12328. doi:10.1111/xen.12328


Anti-Apoptotic Effects of HO1 And A20 Expression in Endothelial Cells of Multi-Transgenic Pigs for Xenotransplantation

Amy S. Dandro1, Carol Phelps1, Todd Vaught1, Stephen P Butler1, Kasinath Kuravi1, Lori Sorrells1, Jeff Monahan1, David Ayares1.

1Revivicor, Inc, Blacksburg, VA, United States

Introduction: HO1 and A20 have known anti-apoptotic and anti-inflammatory effects therefore there is interest in expressing them in transgenic pigs for xenotransplantation.  Multi-transgenic pigs on a GTKO/CD46 genetic background were produced which expressed HO1 or A20. In this study in-vitro assays were optimized and utilized to determine whether the expression of these transgenes in porcine endothelial cells from multi-transgenic pigs has a functional effect in reducing apoptosis.

Materials and Methods: Porcine aortic endothelial cells (PAEC) were isolated from a GTKO/CD46 pig as well as from GTKO/CD46 pigs expressing human HO1 or A20 transgenes under control of a constitutive CAG promoter, and additionally containing 3 other transgenes including human thrombomodulin (hTBM), endothelial protein C receptor (EPCR), CD47, TFPI or CD39. The 6GE genotypes tested included GTKO/CD46 in combination with: i) TBM.CD39.A20.CD47, ii) TBM.TFPI.EPCR.HO1 or iii) TBM.CD47.EPCR.HO1 where the multigene vector was targeted to the alpha-Gal locus rather than randomly integrated. Expression of the HO1 and A20 transgenes was confirmed by Western blot and flow cytometry, and in pig tissues by immunohistochemistry. PAEC were seeded in 96 well plates and grown to confluency. Staurosporine (STS, 1.0 µM) or hTNF-α (20 ng/mL) was used to stimulate apoptosis. Initial studies determined the appropriate time for detection of caspase as a marker of apoptosis. At that time a Caspase-Glo3/7™ assay was performed. Results from the multi-transgenic pig lines were compared to the GTKO/CD46 line.

Results: Visual cell morphology effects of STS on PAEC were seen almost immediately, however, an increase in caspase level was not detected until approximately 4 hours post treatment.  At 8 hours, differences in caspase between the HO1 and A20 multi-transgenic lines and the GTKO/CD46 control line were evident, therefore an 8 hour assay time was utilized. Visual effects on cell morphology were minimal at 8 hours following hTNF-α stimulation, therefore, the caspase assay was performed 24 hours post treatment on hTNF-α stimulated PAEC. Anti-apoptotic benefits of HO1 and A20 transgene expression were similar regardless of the apoptosis stimulation method used.  A20 and HO1 expressing multi-transgenic lines had significantly less caspase than the GTKO/CD46 control line. HO1 targeted to the alpha-Gal locus resulted in significantly less caspase than random integration of either HO1 or A20 in this limited dataset.

Conclusions: A20 and HO1 transgenes can be expressed in multi-transgenic pigs and have functional effects in reducing apoptosis.  Targeted integration of these genes into the alpha-Gal locus may lead to improved functionality compared to random integration.  Further studies including testing organs from similar multi-transgenic pigs in non-human primates are in progress.

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