Gene Editing in Murine Hematopoietic Stem Cells Using a Novel Ex Vivo Expansion System

概要

［課程－２］
審査の結果の要旨
氏名ベッカー ハンス 次郎
This thesis examines the potential of ex vivo expansion of hematopoietic stem cells (HSCs)
in conjunction with CRISPR/Cas9 gene editing to generate large quantities of bona fide,
genetically modified HSCs for stem cell transplantation. In murine transplantation models,
it is shown that:
1. Using CRISPR/Cas9 gene editing, the PrkdcSCID mutation in the CB17/SCID murine
immunodeficiency model can be corrected in a relevant fraction of HSCs. The edited bulk
population can be expanded ex vivo using a serum-free, synthetic polymer-based medium,
producing a large population of HSCs harboring the corrected Prkdc gene. Upon
transplantation, edited HSCs give rise to B and T lymphocytes which demonstrate a functional
restoration of immunity in vivo.
2. The pan-leukocyte antigen CD45.2, expressed on HSCs from C57BL/6 mice, can efficiently
be converted to the CD45.1 allele with CRISPR/Cas9. Using a novel synthetic polymer,
Soluplus, single-cell cloned CD45.1+ HSCs can be expanded while preserving the expression of
phenotypic stemness markers. After transplantation, clonally expanded, edited HSCs display
self-renewal and differentiation characteristics. Peripheral blood chimerism correlated with
the fraction of CD201(EPCR)+ and CD150+ double positive cells in the expanded population.
3. Following these results, the single cell expansion approach was applied to the SCID model.
Bulk edited SCID HSCs were cloned and colonies were screened for high expression of CD201
and CD150 as well as for the presence of a corrected Prkdc allele by means of direct sequencing.
Selected clones were pooled and transplanted into CB17/SCID recipients, which subsequently
showed reconstitution of B and T cell compartments.
This work demonstrates that gene editing of HSCs can restore immunity in SCID mice on a
molecular and functional level. Furthermore, a polymer-based culture system permits single
cell expansion of HSCs and direct sequencing, enabling marker-free selection of clones with
the desired modifications. Engraftment of these clones can be estimated by quantifying the
expression of HSC stemness markers. These results significantly contribute to the field of
genome editing and has major potential for adoption in hematopoietic gene therapy.
よって本論文は博士（ 医学 ）の学位請求論文として合格と認められる。