Palmitoylated phosphodiester gapmer designs with albumin binding capacity and maintained in vitro gene silencing activity

Yunpeng Cai, Anna-Marie Makarova, Jesper Wengel, Kenneth A Howard

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Abstract

BACKGROUND: Antisense gapmer oligonucleotide drugs require delivery and biodistribution enabling technologies to increase in vivo efficacy. An attractive approach is their binding and consequent transport by the endogenous human serum albumin pool as mediated by fatty acid incorporation into the gapmer design.

METHODS: The present study investigated the effect of palmitoyl modification and position on albumin-binding, cellular uptake and in vitro gene silencing of gapmers with either a phosphorothioate (PS) or phosphodiester (PO) backbone.

RESULTS: Two palmitoyls positioned exclusively at the 5' end, or a single palmitoyl at both the 3' and 5' positions, showed similar binding to human serum albumin as demonstrated by a gel-shift assay. Decreased cellular uptake determined by flow cytometry (27% compared to nonpalmitoyl gapmers) was observed for palmitoylated Cy5.5 labelled gapmers. However, HER3 (human epidermal growth factor receptor 3) gene silencing was exhibited by the palmitoylated gapmers with transfection agent in PC-3 and Caco-2 cells (68% and 62%, respectively), which was comparable to nonpalmitoyl gapmers (68% and 82%, respectively). Importantly, PO gapmers with a single palmitoyl positioned at both the 3' and 5' positions showed high silencing efficiencies (68% and 66% in PC-3 and Caco-2 cells, respectively) similar to those of PS nonpalmitoylated gapmers (67% and 66% in PC-3 and Caco-2 cells, respectively) in the absence of a transfection agent.

CONCLUSIONS: The present study defines phosphodiester gapmer design criteria exhibiting high gene silencing activity and albumin binding that may be utilized with potentially less in vivo toxicity that can be associated with phosphorothioate gapmer designs.

Original languageEnglish
JournalJournal of Gene Medicine
Volume20
Issue number7-8
Pages (from-to)1-6
ISSN1099-498X
DOIs
Publication statusPublished - 2018

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Caco-2 Cells
Albumins
erbB-1 Genes
Flow Cytometry
Fatty Acids
Pharmaceutical Preparations
In Vitro Techniques

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@article{8f9cf00e2e5d4ed79901609d599fa59f,
title = "Palmitoylated phosphodiester gapmer designs with albumin binding capacity and maintained in vitro gene silencing activity",
abstract = "BACKGROUND: Antisense gapmer oligonucleotide drugs require delivery and biodistribution enabling technologies to increase in vivo efficacy. An attractive approach is their binding and consequent transport by the endogenous human serum albumin pool as mediated by fatty acid incorporation into the gapmer design.METHODS: The present study investigated the effect of palmitoyl modification and position on albumin-binding, cellular uptake and in vitro gene silencing of gapmers with either a phosphorothioate (PS) or phosphodiester (PO) backbone.RESULTS: Two palmitoyls positioned exclusively at the 5' end, or a single palmitoyl at both the 3' and 5' positions, showed similar binding to human serum albumin as demonstrated by a gel-shift assay. Decreased cellular uptake determined by flow cytometry (27{\%} compared to nonpalmitoyl gapmers) was observed for palmitoylated Cy5.5 labelled gapmers. However, HER3 (human epidermal growth factor receptor 3) gene silencing was exhibited by the palmitoylated gapmers with transfection agent in PC-3 and Caco-2 cells (68{\%} and 62{\%}, respectively), which was comparable to nonpalmitoyl gapmers (68{\%} and 82{\%}, respectively). Importantly, PO gapmers with a single palmitoyl positioned at both the 3' and 5' positions showed high silencing efficiencies (68{\%} and 66{\%} in PC-3 and Caco-2 cells, respectively) similar to those of PS nonpalmitoylated gapmers (67{\%} and 66{\%} in PC-3 and Caco-2 cells, respectively) in the absence of a transfection agent.CONCLUSIONS: The present study defines phosphodiester gapmer design criteria exhibiting high gene silencing activity and albumin binding that may be utilized with potentially less in vivo toxicity that can be associated with phosphorothioate gapmer designs.",
author = "Yunpeng Cai and Anna-Marie Makarova and Jesper Wengel and Howard, {Kenneth A}",
note = "{\circledC} 2018 John Wiley & Sons, Ltd.",
year = "2018",
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language = "English",
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pages = "1--6",
journal = "Journal of Gene Medicine",
issn = "1099-498X",
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Palmitoylated phosphodiester gapmer designs with albumin binding capacity and maintained in vitro gene silencing activity. / Cai, Yunpeng; Makarova, Anna-Marie; Wengel, Jesper; Howard, Kenneth A.

In: Journal of Gene Medicine, Vol. 20, No. 7-8, 2018, p. 1-6.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Palmitoylated phosphodiester gapmer designs with albumin binding capacity and maintained in vitro gene silencing activity

AU - Cai, Yunpeng

AU - Makarova, Anna-Marie

AU - Wengel, Jesper

AU - Howard, Kenneth A

N1 - © 2018 John Wiley & Sons, Ltd.

PY - 2018

Y1 - 2018

N2 - BACKGROUND: Antisense gapmer oligonucleotide drugs require delivery and biodistribution enabling technologies to increase in vivo efficacy. An attractive approach is their binding and consequent transport by the endogenous human serum albumin pool as mediated by fatty acid incorporation into the gapmer design.METHODS: The present study investigated the effect of palmitoyl modification and position on albumin-binding, cellular uptake and in vitro gene silencing of gapmers with either a phosphorothioate (PS) or phosphodiester (PO) backbone.RESULTS: Two palmitoyls positioned exclusively at the 5' end, or a single palmitoyl at both the 3' and 5' positions, showed similar binding to human serum albumin as demonstrated by a gel-shift assay. Decreased cellular uptake determined by flow cytometry (27% compared to nonpalmitoyl gapmers) was observed for palmitoylated Cy5.5 labelled gapmers. However, HER3 (human epidermal growth factor receptor 3) gene silencing was exhibited by the palmitoylated gapmers with transfection agent in PC-3 and Caco-2 cells (68% and 62%, respectively), which was comparable to nonpalmitoyl gapmers (68% and 82%, respectively). Importantly, PO gapmers with a single palmitoyl positioned at both the 3' and 5' positions showed high silencing efficiencies (68% and 66% in PC-3 and Caco-2 cells, respectively) similar to those of PS nonpalmitoylated gapmers (67% and 66% in PC-3 and Caco-2 cells, respectively) in the absence of a transfection agent.CONCLUSIONS: The present study defines phosphodiester gapmer design criteria exhibiting high gene silencing activity and albumin binding that may be utilized with potentially less in vivo toxicity that can be associated with phosphorothioate gapmer designs.

AB - BACKGROUND: Antisense gapmer oligonucleotide drugs require delivery and biodistribution enabling technologies to increase in vivo efficacy. An attractive approach is their binding and consequent transport by the endogenous human serum albumin pool as mediated by fatty acid incorporation into the gapmer design.METHODS: The present study investigated the effect of palmitoyl modification and position on albumin-binding, cellular uptake and in vitro gene silencing of gapmers with either a phosphorothioate (PS) or phosphodiester (PO) backbone.RESULTS: Two palmitoyls positioned exclusively at the 5' end, or a single palmitoyl at both the 3' and 5' positions, showed similar binding to human serum albumin as demonstrated by a gel-shift assay. Decreased cellular uptake determined by flow cytometry (27% compared to nonpalmitoyl gapmers) was observed for palmitoylated Cy5.5 labelled gapmers. However, HER3 (human epidermal growth factor receptor 3) gene silencing was exhibited by the palmitoylated gapmers with transfection agent in PC-3 and Caco-2 cells (68% and 62%, respectively), which was comparable to nonpalmitoyl gapmers (68% and 82%, respectively). Importantly, PO gapmers with a single palmitoyl positioned at both the 3' and 5' positions showed high silencing efficiencies (68% and 66% in PC-3 and Caco-2 cells, respectively) similar to those of PS nonpalmitoylated gapmers (67% and 66% in PC-3 and Caco-2 cells, respectively) in the absence of a transfection agent.CONCLUSIONS: The present study defines phosphodiester gapmer design criteria exhibiting high gene silencing activity and albumin binding that may be utilized with potentially less in vivo toxicity that can be associated with phosphorothioate gapmer designs.

U2 - 10.1002/jgm.3025

DO - 10.1002/jgm.3025

M3 - Journal article

C2 - 29800498

VL - 20

SP - 1

EP - 6

JO - Journal of Gene Medicine

JF - Journal of Gene Medicine

SN - 1099-498X

IS - 7-8

ER -