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Decellularized pig pulmonary heart valves—Depletion of nucleic acids measured by proviral PERV pol
Author(s) -
Godehardt Antonia W.,
Ramm Robert,
Gulich Barbara,
Tönjes Ralf R.,
Hilfiker Andres
Publication year - 2020
Publication title -
xenotransplantation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.052
H-Index - 61
eISSN - 1399-3089
pISSN - 0908-665X
DOI - 10.1111/xen.12565
Subject(s) - decellularization , nuclease , nucleic acid , virology , dna , chemistry , biology , microbiology and biotechnology , biochemistry , extracellular matrix
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.