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MENDIOLA PLA ET AL.
In 2021, the Food and Drug Administration (FDA)
convened on the safety of AAV-based gene therapies and
discussed the lethal risks associated with off-target expression.27
The most common adverse effect is hepatotoxicity,
driven by cytotoxic T cell responses to transduced
liver cells. This complication is largely associated with
systemically administered rAAV vectors.28 We did not
observe any evidence of liver transduction or liver injury
in this study, highlighting a major advantage of ex vivo
AAV delivery. Additionally, utility of ex vivo AAV delivery
facilitates the delivery of viral vector doses resulting
in high vector genomes in transduced cells of the organ. It
remains to be examined what potential consequences can
result from this following ex vivo AAV delivery. Following
systemic AAV administration, high VGCs per cell
can cause immunogenic complications due to activation of
innate immune responses against the viral vector and lead
to activation of adaptive immune responses.28,29
In these settings, doses are typically on the order of
1013-1014 VGC/kg weight of the recipient. Considering
this, the doses utilized in this study ranged from 6.25 · 1011
to 2.38 · 1012 VGC/kg weight of the recipient, which is
substantially lower. Additionally, the animals in our experimental
design received standard triple immunosuppression
therapy for organ transplantation throughout the
study duration, which would have blunted any potential
immune response.
This study also brings to the forefront that AAV
transduction can be enhanced by the composition of the
perfusate during EVMP delivery. The transduction enhancement
of the perfusate and perfusate components was
observed in several different cell types, and in particular
with SASTG. Enhancement in SASTG transduction was
described in Messina et al. where addition of heparin alone
during transduction of911 cells increased rAAV-mediated
luciferase expression.25 In the current study, the addition
of methylprednisolone, a component ofOCS perfusate and
standard immunosuppression agent in transplantation,
consistently increased the transduction efficacy ofSASTG
across multiple cell lines. Transduction enhancement of
rAAVs with the addition of steroids has also been reported
in other studies.30,31 Calcium gluconate has also been
described to enhance transduction of Ad vectors in noncardiac
cell lines.32
In this study, we note its enhancing effect in SASTG
transduction of RNCMs. While results from cell-based
assays may not always reflect what occurs in in vivo AAV
transduction, we utilized these results to approximately
understand what effects on AAV transduction to expect,
particularly that transduction be consistently enhanced
across different cell lines. A limitation of this is that the
results from the cell-based analyses may not directly reflect
the results we present from the AAV transduced porcine
hearts. Further characterization of transduction enhancing
factors that can be used to optimize perfusion solutions for
gene therapies in transplantation is imperative to maximize
the efficiency of AAV-mediated gene delivery and improve
the clinical practicality of these interventions.
CONCLUSIONS
Robust transgene expression can be achieved in a dosedependent
manner in porcine cardiac allografts when
rAAV is administered during normothermic EVMP. Offtarget
transgene expression was not seen and the allografts
did not display any inflammatory response to the viral
vector treatment. Future directions will utilize this method
to deliver therapeutic transgenes to prevent negative recipient
immune responses to the allografts.
ACKNOWLEDGMENTS
The authors thank TransMedics, Inc., for providing financial
support and generously donating the OCS supplies
to conduct these experiments. They thank the Duke Division
of Cardiothoracic Surgery for providing substantial financial
support to conduct these experiments. They also thank
the members of the Duke Division of Laboratory Animal
Resources for providing significant support during the surgeries
and follow-up care for the animals in this study. Finally,
the authors thank the members of Duke Perfusion
Services for their generous support during the surgeries.
AUTHORS' CONTRIBUTIONS
D.E.B., C.A.M., Y.C., and M.M.P. designed the study.
M.M.P., Y.C., A.R., F.H.L., M.F.S., R.T.G., J.N.R., M.B.,
A.E., C.A.M., andD.E.B. performed the experiments. C.W.,
L.E.G., S.H., J.N.S., and P.L. provided valuable advice and
supplies. M.M.P., Y.C., C.G., C.A.M., and D.E.B. analyzed
the data. All authors contributed to writing the article.
AUTHOR DISCLOSURE
P.L. is an employee of TransMedics, Inc., Carmelo
Milano received a financial gift from TransMedics, Inc., to
fund the experiments. All other authors have no disclosures
to declare.
FUNDING INFORMATION
Funding was provided by the Duke Division of Cardiothoracic
Surgery and TransMedics, Inc. M.M.P. is supported
by T32HL007101.
SUPPLEMENTARY MATERIAL
Supplementary Table S1
Supplementary Table S2
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
Supplementary Figure S4
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