TY - JOUR

T1 - Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

AU - Ponomartsev, Sergey V.

AU - Sinenko, Sergey A.

AU - Skvortsova, Elena V.

AU - Liskovykh, Mikhail A.

AU - Voropaev, Ivan N.

AU - Savina, Maria M.

AU - Kuzmin, Andrey A.

AU - Kuzmina, Elena Yu

AU - Kondrashkina, Alexandra M.

AU - Larionov, Vladimir

AU - Kouprina, Natalay

AU - Tomilin, Alexey N.

N1 - Copyright: This record is sourced from MEDLINE/PubMed, a database of the U.S. National Library of Medicine

PY - 2020/4/3

Y1 - 2020/4/3

N2 - Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/- mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/- iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector-the alphoidtetO-HAC.

AB - Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/- mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/- iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector-the alphoidtetO-HAC.

KW - alphoidtetO-HAC

KW - cell reprogramming

KW - coagulation factor VIII

KW - hemophilia

KW - human artificial chromosome (HAC)

KW - induced pluripotent stem cells (iPSCs)

KW - microcell-mediated chromosome transfer (MMCT)

KW - CELLS

KW - INTEGRATION-FREE

KW - HAC

KW - GENOMICS

KW - alphoid(tetO)-HAC

KW - DNA

KW - CONSTRUCTION

KW - TRANSFORMATION-ASSOCIATED RECOMBINATION

KW - GENERATION

KW - CONDITIONAL CENTROMERE

KW - EXPRESSION

UR - http://www.scopus.com/inward/record.url?scp=85090098719&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/a8559bc6-104c-3d57-af26-e1cc0eaf4b61/

U2 - 10.3390/cells9040879

DO - 10.3390/cells9040879

M3 - Article

C2 - 32260189

AN - SCOPUS:85090098719

VL - 9

JO - Cells

JF - Cells

SN - 2073-4409

IS - 4

M1 - 879

ER -