RT Journal Article
SR Electronic
T1 Longitudinal clonal tracking in humanized mice reveals sustained polyclonal repopulation of gene-modified human-HSPC despite vector integration bias
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.08.21.261537
DO 10.1101/2020.08.21.261537
A1 Gajendra W. Suryawanshi
A1 Hubert Arokium
A1 Sanggu Kim
A1 Wannisa Khamaikawin
A1 Samantha Lin
A1 Saki Shimizu
A1 Koollawat Chupradit
A1 YooJin Lee
A1 Yiming Xie
A1 Xin Guan
A1 Vasantika Suryawanshi
A1 Angela P. Presson
A1 Dong-Sung An
A1 Irvin S. Y. Chen
YR 2021
UL http://biorxiv.org/content/early/2021/07/19/2020.08.21.261537.abstract
AB Background Current understanding of hematopoiesis is largely derived from mouse models that are physiologically distant from humans. Humanized mice provide the most physiologically relevant small animal model to study human diseases, most notably preclinical gene-therapy studies. However, the clonal repopulation dynamics of human hematopoietic stem and progenitor cells (HSPC) in these animal models is only partially understood. Using a new clonal tracking methodology designed for small sample volumes, we aim to reveal the underlying clonal dynamics of human cell repopulation in a mouse environment.Methods Humanized BLT (bone marrow-liver-thymus) mice were generated by transplanting lentiviral vector transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25µl of mouse blood for longitudinal and quantitative clonal analysis of human HSPC repopulation in mouse environment. We explored transcriptional and epigenetic features of human HSPC for possible VIS bias.Results 897 HSPC clones were longitudinally tracked in BLT mice—providing a first-ever demonstration of clonal dynamics and competitive expansion of therapeutic and control vector-modified human cell populations simultaneously repopulating in the same humanized mice. The polyclonal repopulation stabilized at 19 weeks post-transplant and the contribution of the largest clone doubled within 4 weeks. Moreover, 550 (∼60%) clones persisted over 6 weeks and were highly shared between different organs. The normal clonal profiles confirmed the safety of our gene therapy vectors. Multi-omics analysis of human FL-HSPC revealed that 54% of vector integrations in repopulating clones occurred within ±1kb of H3K36me3-enriched regions.Conclusions Human repopulation in mice is polyclonal and stabilizes more rapidly than that previously observed in humans. VIS preference for H3K36me3 has no apparent negative effects on HSPC repopulation. Our study provides a methodology to longitudinally track clonal repopulation in small animal models extensively used for stem cell and gene-therapy research and with lentiviral vectors designed for clinical applications. Results of this study provide a framework for understanding the clonal behavior of human HPSC repopulating in a mouse environment, critical for translating results from humanized mice models to the human settings.Competing Interest StatementDr. Irvin S.Y. Chen has a financial interest in CSL Behring and Calimmune Inc. No funding was provided by these companies to support this work; Dr. Dong Sung An has a financial interest in Calimmune Inc and CSL Behring that the University of California Regents have licensed intellectual property invented by Dong Sung An, that is being used in the research, to Calimmune Inc. No funding was provided by these companies to support this work. All other authors declare no competing interests.HSPChematopoietic stem and progenitor cellsLoVIS-Seqlow volume vector integration site sequencingBLTBone marrow-liver-thymusFLfetal liverMDAMultiple displacement amplification.