Friday October 11, 2019 from 08:00 to 08:30
Droplet digital PCR methods for characterizing porcine endogenous retrovirus transmission of organ specific cells from PERV-C free cloned piglets
Zheng-Yu Wang1, Juliet Easlick1, Jose Estrada1, Luz Reyes1, Gregory Martens1, Joseph Ladowski1, Edward Gray1, Devin Eckhoff1, Mathew Tector1, Joseph Tector1.
1Transplant Division of Surgery, University of Alabama at Birmingham, School of Medicine, Birmingham, AL, United States
Background: Xenotransplantation addresses a potential solution to the donor organ shortage. However, there are some concerns about recipient safety because the risk of porcine endogenous retrovirus (PERV) transmission to human cells remains unknown. PERV-A and -B are integrated in the genome of all pigs, but PERV-C is not in all pigs. In this study, the aim was to apply droplet digital PCR (ddPCR) methods for characterizing PERV transmission of organ specific cells from PERV-C free cloned pigs using both HEK293 co-incubation system and polybrene-enhanced infection system.
Methods: PERV-C free Pigs were selected and purchased from Prestage Farms of Mississippi, Inc. Three PERV-C free cloned piglets were generated using somatic nuclear transform technology. Human embryonic kidney 293 (HEK293), 293-PERV-PK-CIRCE (293-PERV) and porcine PK15 were purchased, respectively from ATTCC and Millipore Sigma. Human PBMC were isolated from healthy people. The followings were used as PERV producer cells including PBMC, renal microvascular endothelial cells (RMEC), liver sinusoidal endothelial cells (LSEC), aortic endothelial cells (AEC), and skin fibroblast cells (SFC) from PERV-C free cloned piglets. ddPCR methods was performed to detect and quantify PERV expression and its reverse transcriptase (RT) function of infected HEK293 using co-incubation system and polybrene-enhanced infection system.
Results: Using the ultrasensitive and precise ddPCR methods, we confirmed that PERV-C were negative in organ specific cells from three cloned piglets following pig selection of negative PERV-C expression, observed that PERV-A and -B copy number of the mitogen-activated PBMC was significantly high, but the copy number was not a significant difference between renal, liver, and aorta endothelial cells, and found that no PERV-A and –B were detected from infected HEK293 using both HEK293 co-incubation system (Figure 1) and polybrene-enhanced infection system. Furthermore MS2-based ddPCR assay for RT activity shows that MS2 copy number of infected HEK293 cell culture supernatant was not significantly changed using both HEK293 co-incubation system (Figure 1) and polybrene-enhanced infection system.
Conclusions: ddPCR-based methods are valuable for developing PERV-C free cloned pigs via screening the lack of PERV-C expression to avoid the generation of high replication recombinants PERV-A/C. Except mitogen-activated PBMC, the expressions of PERV were not significantly difference between organ-specific cells from PERV-C free piglets. No transmission of PERV from organ-specific donor cells was observed to human target cells including 293T, which are infected easily by PERV-A and –B, suggesting that PERV-C free pigs might be used as potential donors for pig-to-human xenotransplantation.
Disclosures: A.J.T. is the founder of Xenobridge LLC, and holds and applied for patents related to xenotransplantation. Portions of this work have been funded by University of Alabama at Birmingham, United Therapeutics. .