# CD47;Rag2;IL-2rγ triple knock-out mice pre-conditioning with busulfan could be a novel platform for generating hematopoietic stem cells engrafted humanized mice

## TL;DR

This study introduces a new mouse model that can be used to create humanized mice with improved long-term human immune system development and reduced disease risks.

## Contribution

The novel RTKO mouse strain with CD47, Rag2, and IL-2rγ knockouts and busulfan pre-conditioning is proposed as an improved platform for humanized mice.

## Key findings

- RTKO mice pretreated with busulfan showed the highest and longest maintenance of human CD45+ cells over 40 weeks.
- Human leukocyte reconstruction, including hCD3, was most prominent in RTKO busulfan-treated mice.
- Only two mice died in all groups, with no life-threatening GvHD except in the deceased mice.

## Abstract

Humanized mouse models to recapitulate human biological systems still have limitations, such as the onset of lethal graft-versus-host disease (GvHD), a variable success rate, and the low accessibility of total body irradiation (TBI). Recently, mice modified with the CD47-SIRPA axis have been studied to improve humanized mouse models. However, such trials have been rarely applied in NOD mice. In this study, we created a novel mouse strain, NOD-CD47nullRag2nullIL-2rγnull (RTKO) mice, and applied it to generate humanized mice.

Four-week-old female NOD-Rag2nullIL-2rγnull (RID) and RTKO mice pre-conditioned with TBI or busulfan (BSF) injection were used for generating human CD34+ hematopoietic stem cell (HSC) engrafted humanized mice. Clinical signs were observed twice a week, and body weight was measured once a week. Flow cytometry for human leukocyte antigens was performed at intervals of four weeks or two weeks, and mice were sacrificed at 48 weeks after HSC injection.

For a long period from 16 to 40 weeks post transplantation, the percentage of hCD45 was mostly maintained above 25% in all groups, and it was sustained the longest and highest in the RTKO BSF group. Reconstruction of human leukocytes, including hCD3, was also most prominent in the RTKO BSF group. Only two mice died before 40 weeks post transplantation in all groups, and there were no life-threatening GvHD lesions except in the dead mice. The occurrence of GvHD has been identified as mainly due to human T cells infiltrating tissues and their related cytokines.

Humanized mouse models under all conditions applied in this study are considered suitable models for long-term experiments based on the improvement of human leukocytes reconstruction and the stable animal health. Especially, RTKO mice pretreated with BSF are expected to be a valuable platform not only for generating humanized mice but also for various immune research fields.