Decellularized pig pulmonary heart valves—Depletion of nucleic acids measured by proviral PERV pol

Abstract

Background: Decellularized human pulmonary heart valve (dhHV) scaffolds have been shown to be the gold standard especially for younger, adolescent patients.However, human heart valves are limited in availability. Xenogeneic decellularized pig heart valves (dpHV) may serve as alternative. Methods:The efficacy of DNA reduction processes upon decellularization of heart valves from German Landrace pigs was analyzed by measurements of remaining nucleic acids including proviral porcine endogenous retrovirus (PERV) sequences.Porcine pulmonary heart valves (pPHV) were decellularized by three different protocols and further treated with DNaseI or Benzonase, at varying incubation times.DNA isolated from valve associated muscle (m), valve cusp (c), and pulmonary artery (pa) was monitored by PCR and qRT-PCR using GAPDH and the PERV polymerase (pol) for read-out.Results: Decellularization of pPHV led to a significant reduction of DNA (>99%) which could be further significantly increased for (m) and (pa) by nuclease treatment, reducing proviral PERV pol from approximately 5 × 10 7 to 5 × 10 3 copies/mg in nuclease treated tissues. Conclusions:Both nucleases demonstrated comparable activities. But DNaseI revealed to be less consistent for PERV, especially at muscular tissue. Noteworthy, remaining proviral sequences are still detectable by PCR; however, due to the absence of the cellular replication machinery the production of infectious particles is not expected. Decellularization and nuclease treatment of pPHV is an efficient procedure to reduce the DNA content including PERV, thus represents a valuable option to increase virus safety independently from the source animal background. K E Y W O R D SPERV, virus safety, xenogeneic decellularized heart valve

Xenotransplantation of decellularized pig heart valves—Regulatory aspects in Europe

“…These valves differ significantly from glutaraldehyde‐fixed bioprostheses being vital matrices, open for recellularization, and the resulting physiologic remodeling of the implant 6,26,27 . Even if undergoing a decellularization procedure, the presence of resilient DNA 27‐29 and cells that survive the decellularization procedures were reported 30‐32 . It was shown that none of the present decellularization methods can achieve 100% cell removal, even if the remaining cells are not vital.…”

Xenotransplantation literature update, March/April 2020

“…They claimed that the function of decellularized xenogeneic heart valves was comparable to that of allogeneic controls. Godehardt et al also considered that decellularization and nuclease treatment of porcine pulmonary heart valves is an efficient procedure to reduce the DNA content including PERV, which may be beneficial to decrease immunogenicity and zoontic risk 23 . Therefore, efficient decellularization combined with enzymatic removal of DNA might be a feasible method to overcome the immunological barrier causing rapid destruction of the xenogeneic valve.…”

Introduction to the theme issue on regulatory aspects of xenotransplantation

“…However, tissues including heart valves are nowadays decellularized by other means, which asks for a separate consideration in regulatory oversight. Noteworthy, this is increasingly relevant because there is increased interest in using decellularized tissue as scaffold in regenerative medicine and proven evidence of the presence of nucleic acid even after treatment: This was, among others, documented recently in a publication by Antonia Godehardt and coworkers in this journal 15 and resulted in a major point of discussion in the session at the IXA Congress mentioned above. After an opinion paper by Joachim Denner on definitions, the regulation of these decellularized tissues by the European Medicines Agency (EMA) is described by Antonia Godehardt and Ralf Tönjes.…”