Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells

Lucas M. Sjeklocha, Chang Won Park, Phillip Y.P. Wong, Mark J. Roney, John D Belcher, Dan S Kaufman, Gregory M Vercellotti, Robert P Hebbel, Clifford J Steer

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34 + cells with DsRed and a hybrid IHK-β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK-β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-β-globin transgene for gene therapy of sickle cell disease.

Original languageEnglish (US)
Article numbere29110
JournalPloS one
Volume6
Issue number12
DOIs
StatePublished - Dec 28 2011

Fingerprint

Beauty
Globins
Hematopoietic Stem Cells
transgenes
Transgenes
sickle cell anemia
gene therapy
Gene therapy
Sickle Cell Anemia
Genetic Therapy
Insulator Elements
Transposases
Messenger RNA
Erythroid Cells
cells
Aptitude
blood cells
K562 Cells
hematopoietic stem cells
transposons

Cite this

Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells. / Sjeklocha, Lucas M.; Park, Chang Won; Wong, Phillip Y.P.; Roney, Mark J.; Belcher, John D; Kaufman, Dan S; Vercellotti, Gregory M; Hebbel, Robert P; Steer, Clifford J.

In: PloS one, Vol. 6, No. 12, e29110, 28.12.2011.

Research output: Contribution to journalArticle

@article{3d9c9d52c4624e8cabb4a8e2b99ccbbf,
title = "Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells",
abstract = "Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34 + cells with DsRed and a hybrid IHK-β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK-β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-β-globin transgene for gene therapy of sickle cell disease.",
author = "Sjeklocha, {Lucas M.} and Park, {Chang Won} and Wong, {Phillip Y.P.} and Roney, {Mark J.} and Belcher, {John D} and Kaufman, {Dan S} and Vercellotti, {Gregory M} and Hebbel, {Robert P} and Steer, {Clifford J}",
year = "2011",
month = "12",
day = "28",
doi = "10.1371/journal.pone.0029110",
language = "English (US)",
volume = "6",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "12",

}

TY - JOUR

T1 - Erythroid-specific expression of β-globin from Sleeping Beauty-transduced human hematopoietic progenitor cells

AU - Sjeklocha, Lucas M.

AU - Park, Chang Won

AU - Wong, Phillip Y.P.

AU - Roney, Mark J.

AU - Belcher, John D

AU - Kaufman, Dan S

AU - Vercellotti, Gregory M

AU - Hebbel, Robert P

AU - Steer, Clifford J

PY - 2011/12/28

Y1 - 2011/12/28

N2 - Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34 + cells with DsRed and a hybrid IHK-β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK-β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-β-globin transgene for gene therapy of sickle cell disease.

AB - Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34 + cells with DsRed and a hybrid IHK-β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK-β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-β-globin transgene for gene therapy of sickle cell disease.

UR - http://www.scopus.com/inward/record.url?scp=84555218905&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84555218905&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0029110

DO - 10.1371/journal.pone.0029110

M3 - Article

VL - 6

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 12

M1 - e29110

ER -