A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy

Muath Bishawi, Jun-Neng Roan, Carmelo A. Milano, Mani A. Daneshmand, Jacob N. Schroder, Yuting Chiang, Franklin H. Lee, Zachary D. Brown, Adam Nevo, Michael J. Watson, Trevelyn Rowell, Sally Paul, Paul Lezberg, Richard Walczak, Dawn E. Bowles

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Abstract

Clinically, both percutaneous and surgical approaches to deliver viral vectors to the heart either have resulted in therapeutically inadequate levels of transgene expression or have raised safety concerns associated with extra-cardiac delivery. Recent developments in the field of normothermic ex vivo cardiac perfusion storage have now created opportunities to overcome these limitations and safety concerns of cardiac gene therapy. This study examined the feasibility of ex vivo perfusion as an approach to deliver a viral vector to a donor heart during storage and the resulting bio distribution and expression levels of the transgene in the recipient post-transplant. The influence of components (proprietary solution, donor blood, and ex vivo circuitry tubing and oxygenators) of the Organ Care System (OC) (TransMedics, Inc., Andover MA) on viral vector transduction was examined using a cell-based luciferase assay. Our ex vivo perfusion strategy, optimized for efficient Adenoviral vector transduction, was utilized to deliver 5 × 1013 total viral particles of an Adenoviral firefly luciferase vector with a cytomegalovirus (CMV) promotor to porcine donor hearts prior to heterotopic implantation. We have evaluated the overall levels of expression, protein activity, as well as the bio distribution of the firefly luciferase protein in a series of three heart transplants at a five-day post-transplant endpoint. The perfusion solution and the ex vivo circuitry did not influence viral vector transduction, but the serum or plasma fractions of the donor blood significantly inhibited viral vector transduction. Thus, subsequent gene delivery experiments to the explanted porcine heart utilized an autologous blood recovery approach to remove undesired plasma or serum components of the donor blood prior to its placement into the circuit. Enzymatic assessment of luciferase activity in tissues (native heart, allograft, liver etc.) obtained post-transplant day five revealed wide-spread and robust luciferase activity in all regions of the allograft (right and left atria, right and left ventricles, coronary arteries) compared to the native recipient heart. Importantly, luciferase activity in recipient heart, liver, lung, spleen, or psoas muscle was within background levels. Similar to luciferase activity, the luciferase protein expression in the allograft appeared uniform and robust across all areas of the myocardium as well as in the coronary arteries. Importantly, despite high copy number of vector genomic DNA in transplanted heart tissue, there was no evidence of vector DNA in either the recipient’s native heart or liver. Overall we demonstrate a simple protocol to achieve substantial, global gene delivery and expression isolated to the cardiac allograft. This introduces a novel method of viral vector delivery that opens the opportunity for biological modification of the allograft prior to implantation that may improve post-transplant outcomes.

Original languageEnglish
Article number8029
JournalScientific reports
Volume9
Issue number1
DOIs
Publication statusPublished - 2019 Dec 1

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Heart Transplantation
Genetic Therapy
Perfusion
Luciferases
Allografts
Blood Donors
Firefly Luciferases
Transplants
Heart Atria
Transgenes
Heart Ventricles
Liver
Coronary Vessels
Swine
Tissue Donors
Psoas Muscles
Oxygenators
Safety
Proteins
DNA

All Science Journal Classification (ASJC) codes

  • General

Cite this

Bishawi, M., Roan, J-N., Milano, C. A., Daneshmand, M. A., Schroder, J. N., Chiang, Y., ... Bowles, D. E. (2019). A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy. Scientific reports, 9(1), [8029]. https://doi.org/10.1038/s41598-019-43737-y
Bishawi, Muath ; Roan, Jun-Neng ; Milano, Carmelo A. ; Daneshmand, Mani A. ; Schroder, Jacob N. ; Chiang, Yuting ; Lee, Franklin H. ; Brown, Zachary D. ; Nevo, Adam ; Watson, Michael J. ; Rowell, Trevelyn ; Paul, Sally ; Lezberg, Paul ; Walczak, Richard ; Bowles, Dawn E. / A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy. In: Scientific reports. 2019 ; Vol. 9, No. 1.
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author = "Muath Bishawi and Jun-Neng Roan and Milano, {Carmelo A.} and Daneshmand, {Mani A.} and Schroder, {Jacob N.} and Yuting Chiang and Lee, {Franklin H.} and Brown, {Zachary D.} and Adam Nevo and Watson, {Michael J.} and Trevelyn Rowell and Sally Paul and Paul Lezberg and Richard Walczak and Bowles, {Dawn E.}",
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Bishawi, M, Roan, J-N, Milano, CA, Daneshmand, MA, Schroder, JN, Chiang, Y, Lee, FH, Brown, ZD, Nevo, A, Watson, MJ, Rowell, T, Paul, S, Lezberg, P, Walczak, R & Bowles, DE 2019, 'A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy', Scientific reports, vol. 9, no. 1, 8029. https://doi.org/10.1038/s41598-019-43737-y

A normothermic ex vivo organ perfusion delivery method for cardiac transplantation gene therapy. / Bishawi, Muath; Roan, Jun-Neng; Milano, Carmelo A.; Daneshmand, Mani A.; Schroder, Jacob N.; Chiang, Yuting; Lee, Franklin H.; Brown, Zachary D.; Nevo, Adam; Watson, Michael J.; Rowell, Trevelyn; Paul, Sally; Lezberg, Paul; Walczak, Richard; Bowles, Dawn E.

In: Scientific reports, Vol. 9, No. 1, 8029, 01.12.2019.

Research output: Contribution to journalArticle

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AU - Bishawi, Muath

AU - Roan, Jun-Neng

AU - Milano, Carmelo A.

AU - Daneshmand, Mani A.

AU - Schroder, Jacob N.

AU - Chiang, Yuting

AU - Lee, Franklin H.

AU - Brown, Zachary D.

AU - Nevo, Adam

AU - Watson, Michael J.

AU - Rowell, Trevelyn

AU - Paul, Sally

AU - Lezberg, Paul

AU - Walczak, Richard

AU - Bowles, Dawn E.

PY - 2019/12/1

Y1 - 2019/12/1

N2 - Clinically, both percutaneous and surgical approaches to deliver viral vectors to the heart either have resulted in therapeutically inadequate levels of transgene expression or have raised safety concerns associated with extra-cardiac delivery. Recent developments in the field of normothermic ex vivo cardiac perfusion storage have now created opportunities to overcome these limitations and safety concerns of cardiac gene therapy. This study examined the feasibility of ex vivo perfusion as an approach to deliver a viral vector to a donor heart during storage and the resulting bio distribution and expression levels of the transgene in the recipient post-transplant. The influence of components (proprietary solution, donor blood, and ex vivo circuitry tubing and oxygenators) of the Organ Care System (OC) (TransMedics, Inc., Andover MA) on viral vector transduction was examined using a cell-based luciferase assay. Our ex vivo perfusion strategy, optimized for efficient Adenoviral vector transduction, was utilized to deliver 5 × 1013 total viral particles of an Adenoviral firefly luciferase vector with a cytomegalovirus (CMV) promotor to porcine donor hearts prior to heterotopic implantation. We have evaluated the overall levels of expression, protein activity, as well as the bio distribution of the firefly luciferase protein in a series of three heart transplants at a five-day post-transplant endpoint. The perfusion solution and the ex vivo circuitry did not influence viral vector transduction, but the serum or plasma fractions of the donor blood significantly inhibited viral vector transduction. Thus, subsequent gene delivery experiments to the explanted porcine heart utilized an autologous blood recovery approach to remove undesired plasma or serum components of the donor blood prior to its placement into the circuit. Enzymatic assessment of luciferase activity in tissues (native heart, allograft, liver etc.) obtained post-transplant day five revealed wide-spread and robust luciferase activity in all regions of the allograft (right and left atria, right and left ventricles, coronary arteries) compared to the native recipient heart. Importantly, luciferase activity in recipient heart, liver, lung, spleen, or psoas muscle was within background levels. Similar to luciferase activity, the luciferase protein expression in the allograft appeared uniform and robust across all areas of the myocardium as well as in the coronary arteries. Importantly, despite high copy number of vector genomic DNA in transplanted heart tissue, there was no evidence of vector DNA in either the recipient’s native heart or liver. Overall we demonstrate a simple protocol to achieve substantial, global gene delivery and expression isolated to the cardiac allograft. This introduces a novel method of viral vector delivery that opens the opportunity for biological modification of the allograft prior to implantation that may improve post-transplant outcomes.

AB - Clinically, both percutaneous and surgical approaches to deliver viral vectors to the heart either have resulted in therapeutically inadequate levels of transgene expression or have raised safety concerns associated with extra-cardiac delivery. Recent developments in the field of normothermic ex vivo cardiac perfusion storage have now created opportunities to overcome these limitations and safety concerns of cardiac gene therapy. This study examined the feasibility of ex vivo perfusion as an approach to deliver a viral vector to a donor heart during storage and the resulting bio distribution and expression levels of the transgene in the recipient post-transplant. The influence of components (proprietary solution, donor blood, and ex vivo circuitry tubing and oxygenators) of the Organ Care System (OC) (TransMedics, Inc., Andover MA) on viral vector transduction was examined using a cell-based luciferase assay. Our ex vivo perfusion strategy, optimized for efficient Adenoviral vector transduction, was utilized to deliver 5 × 1013 total viral particles of an Adenoviral firefly luciferase vector with a cytomegalovirus (CMV) promotor to porcine donor hearts prior to heterotopic implantation. We have evaluated the overall levels of expression, protein activity, as well as the bio distribution of the firefly luciferase protein in a series of three heart transplants at a five-day post-transplant endpoint. The perfusion solution and the ex vivo circuitry did not influence viral vector transduction, but the serum or plasma fractions of the donor blood significantly inhibited viral vector transduction. Thus, subsequent gene delivery experiments to the explanted porcine heart utilized an autologous blood recovery approach to remove undesired plasma or serum components of the donor blood prior to its placement into the circuit. Enzymatic assessment of luciferase activity in tissues (native heart, allograft, liver etc.) obtained post-transplant day five revealed wide-spread and robust luciferase activity in all regions of the allograft (right and left atria, right and left ventricles, coronary arteries) compared to the native recipient heart. Importantly, luciferase activity in recipient heart, liver, lung, spleen, or psoas muscle was within background levels. Similar to luciferase activity, the luciferase protein expression in the allograft appeared uniform and robust across all areas of the myocardium as well as in the coronary arteries. Importantly, despite high copy number of vector genomic DNA in transplanted heart tissue, there was no evidence of vector DNA in either the recipient’s native heart or liver. Overall we demonstrate a simple protocol to achieve substantial, global gene delivery and expression isolated to the cardiac allograft. This introduces a novel method of viral vector delivery that opens the opportunity for biological modification of the allograft prior to implantation that may improve post-transplant outcomes.

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