Longitudinal clonal tracking in humanized mice reveals sustained polyclonal repopulation of gene-modified human-HSPC despite vector integration bias

Gajendra W Suryawanshi; Hubert Arokium; Sanggu Kim; Wannisa Khamaikawin; Samantha Lin; Saki Shimizu; Koollawat Chupradit; YooJin Lee; Yiming Xie; Xin Guan; Vasantika Suryawanshi; Angela P Presson; Dong-Sung An; Irvin S Y Chen

doi:10.1186/s13287-021-02601-5

Longitudinal clonal tracking in humanized mice reveals sustained polyclonal repopulation of gene-modified human-HSPC despite vector integration bias

Stem Cell Res Ther. 2021 Oct 7;12(1):528. doi: 10.1186/s13287-021-02601-5.

Authors

Gajendra W Suryawanshi^{1

2}, Hubert Arokium^{1

2}, Sanggu Kim^{3

4

5}, Wannisa Khamaikawin^{6

7}, Samantha Lin⁶, Saki Shimizu⁶, Koollawat Chupradit⁶, YooJin Lee^{1

2}, Yiming Xie^{1

2}, Xin Guan^{1

2}, Vasantika Suryawanshi⁸, Angela P Presson^{9

10}, Dong-Sung An^{2

6}, Irvin S Y Chen^{11

12

13}

Affiliations

¹ Department of Microbiology, Immunology and Molecular Genetics, University of California, 615 Charles E. Young Dr. South, BSRB, Rm 173, Los Angeles, CA, 90095, USA.
² UCLA AIDS Institute, Los Angeles, CA, 90095, USA.
³ Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, 43210, USA.
⁴ Center for Retrovirus Research, The Ohio State University, Columbus, OH, 43210, USA.
⁵ Infectious Disease Institute, The Ohio State University, Columbus, OH, 43210, USA.
⁶ School of Nursing, University of California, Los Angeles, CA, 90095, USA.
⁷ Faculty of Medicine, King Mongkut's Institute of Technology Ladkrabang, Bangkok, 10520, Thailand.
⁸ Department of Molecular and Computational Biology, University of Southern California, Los Angeles, CA, 90089, USA.
⁹ Division of Epidemiology, Department of Internal Medicine, University of Utah, Salt Lake City, 84108, USA.
¹⁰ Department of Biostatistics, University of California, Los Angeles, 90095, USA.
¹¹ Department of Microbiology, Immunology and Molecular Genetics, University of California, 615 Charles E. Young Dr. South, BSRB, Rm 173, Los Angeles, CA, 90095, USA. syuchen@mednet.ucla.edu.
¹² UCLA AIDS Institute, Los Angeles, CA, 90095, USA. syuchen@mednet.ucla.edu.
¹³ Division of Hematology-Oncology, David Geffen School of Medicine at UCLA, Los Angeles, CA, 90095, USA. syuchen@mednet.ucla.edu.

Abstract

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 bone marrow-liver-thymus (hu-BLT) mice were generated by transplanting lentiviral vector-transduced human fetal liver HSPC (FL-HSPC) in NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice implanted with a piece of human fetal thymus. We developed a methodology to track vector integration sites (VIS) in a mere 25 µl 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: A total of 897 HSPC clones were longitudinally tracked in hu-BLT mice-providing a first-ever demonstration of clonal dynamics and coordinated 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 ± 1 kb 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.

Keywords: Clonal dynamics; Gene therapy; HSPC; Humanized mouse; LoVIS-Seq; MDA; VIS tracking.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Disease Models, Animal
Hematopoiesis
Hematopoietic Stem Cell Transplantation*
Hematopoietic Stem Cells*
Humans
Mice
Mice, Inbred NOD
Mice, SCID

Abstract

Publication types

MeSH terms

Grants and funding