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Abstract

The NCG triple immunodeficient mice on a NOD/Nju background lack functional/mature T, B, and NK cells, and have reduced macrophage and dendritic cell function. This study characterized the NCG mouse model for toxicity, engraftment and tumorigenicity assessments of cell therapies, using CD34⁺ hHSPC adult mobilized cells with two myeloablation regimens.

Mice received sub-lethal irradiation or busulfan and were then injected intravenously with CD34⁺ hHSPCs (1.0 x 106 cells/mouse) or PBS (control), while positive control animals received 2 x 106 HL-60 cells/mouse. hCD34+ cell donors were treated with the mobilizing agent G-CSF prior to leukapheresis. Following injections, mouse blood samples were collected to assess engraftment rates by flow cytometry with body weights recorded periodically up to 20 weeks post-cell injection. No significant clinical signs or body weight changes were observed. At week 10 post-cell injection, the peripheral blood chimerism of hCD45+ cells was above 20%. While mCD45+ concentration was constant between week 10 and 17 in whole blood samples, hCD45+ concentration and chimerism slightly decreased at week 17. However, chimerism remained above 10%, with busulfan-treated mice presenting higher values. Chimerism was further assessed by quantifying human Alu sequences in blood and multiple organs using qPCR. Alu sequences were most abundant in the spleen and bone marrow, while lowest in the testes. In the positive control group, expected mortalities due to tumorigenesis were observed between days 27 and 40 post-cell injection. Overall, study results may be used to inform study design and potential toxicological endpoints relevant to non-clinical cell therapy development.

Keywords

NCG mice humanized mice cell therapy hematopoietic stem cells

Introduction

Cell therapy is a rapidly growing therapeutic modality ranging across multiple indications, with applications in treating cancer, infections, allogenic transplantation, and treating autoimmune disorders among other ailments.¹ For example, chimeric antigen receptor (CAR) cell therapy clinical trials increased 4-fold between 2014 and 2018.² Then between 2017 and 2019,³ cell therapies had the highest number of new drugs added to the pharmaceutical industry pipeline, when compared to other common cancer drug classes. Furthermore, the recent FDA approval of CAR-T cell therapy for hematological malignancies in 2017 has only increased the pre-clinical need for assessing these and other cell therapies. In 2019, a press release from the FDA correctly estimated at least 200 investigational new drug (IND) applications in addition to the 800 cell-based or gene therapy INDs in 2019.⁴ Furthermore, the FDA predicted that by 2025, they would approve 10 to 20 cell and gene therapy products per year according to the pharmaceutical industry pipeline and clinical success rates observed in this class.⁵ In response to this rapid development, the FDA increased allocation of resources to support cell and gene therapy studies. This increase in IND applications for cell therapies also reflects the growing need for pre-clinical models for assessing safety and efficacy of cell therapies.

Across the vast therapeutic indications that cell therapies apply to, a major challenge is addressing the complexity of the human immune system and the consequential variability in patient responses to treatments.⁶ Safety concerns pertaining to allogenic CAR-T therapies will differ compared to autologous CAR-T safety assessments. In the former, graft versus host disease (GvHD) is a significant concern, while in the latter, GvHD is less of a concern due to developmental thymic negative selection leading to tolerance.⁷ Similarly, for regenerative medicine indications, an important safety assessment relates to tumorigenicity. It is common practice to conduct non-clinical studies that combine a range of objectives including efficacy and biodistribution but also general toxicity and tumorigenicity.^6,8 A relatively limited number of animal models were characterized to assess cell therapies pre-clinically, such as the BALB/c SCID⁹ or the NOD immunodeficient mouse strains that lack the IL-2r γ chain. Some of the mouse strains used in cell therapy studies include the BALB/c-Rag2 null IL2rγ null or the NSG (NOD/SCID/IL-2Rγ-null). Other mouse models that are used include the NSGW41 (NOD/SCID Il2rg−/− KitW41/W41) that harbor an additional mutant kit receptor decreasing the competition with human HPSCs,¹⁰ and the NBSGW (NOD.Cg-KitW−41J Tyr⁺ Prkdcscid Il2rgtm1Wji/Thom) which can support human cell engraftment without conditioning or myeloablation.¹¹ The current needs in this field have provided impetus for designing new, alternative animal models in order to improve safety assessment of cell therapies.

The objective of the study was to characterize the NCG mouse model as a pre-clinical model to assess toxicity, engraftment, biodistribution, and tumorigenicity for the development of cell therapies. NCG mice are triple immunodeficient with a NOD/Nju background and CRISPR/Cas9 edited genes Prkdc and Il2rg. Consequently, these mice lack functional T, B, and NK cells and have reduced macrophage and dendritic cell function. Importantly, previous work demonstrated that NCG mice were a stable host for human immune cells and tumors in various disease contexts including bladder, colon, and breast cancer.^12-15

The present study consisted of two phases, the first phase optimized total body irradiation dose for myeloablation in NCG mice, while the second phase characterized the model for toxicity, engraftment, biodistribution, and tumorigenicity endpoints with different conditioning regimens. In Phase 1, the optimized irradiation dose was established to be 200 cGy. This dose was well tolerated with no mortalities, clinical observations or changes in body weight observed. In Phase 2, in order to condition the NCG mice for receiving CD34⁺ human hematopoietic stem and progenitor cells (hHSPCs), two types of myeloablation were employed, namely, total body irradiation at 200 cGy or intra-peritoneal busulfan administration. Notably, both of these myeloablation strategies have been investigated for hematopoietic cell transplantation in patients.¹⁶ Mice were then administered a slow bolus intravenous injection of adult mobilized CD34⁺ hHSPCs as a cell product. As a conditioning, the donors providing the hHSPCs were treated with granulocyte colony-stimulating factor (G-CSF) in order to increase (mobilize) peripheral blood CD34⁺ hHSPCs.¹⁷ As a positive control, selected animals received an injection of the tumor cell line HL-60 following irradiation.

Methods

Animals

NCG triple-immunodeficient mice (Mus musculus, NOD-Prkdc^em26Cd52 Il2rg^em26Cd22/NjuCrl) were co-developed by Nanjing Galaxy Biopharma in 2014 and transferred to Charles River in 2016. This model was created by simultaneous CRISPR/Cas9 editing of the Prkdc and Il2rg loci in the NOD/Nju mouse, generating a mouse coisogenic to the NOD/Nju. A 52-base-pair deletion in exon 37 (starting in codon 1619) was created in the Prkdc gene and a 22-base-pair deletion in exon 3 (codon 156) was created in the Il2rg gene. Both male and female NCG mice are homozygous for the Prkdc deletion, while female mice are homozygous and males are hemizygous for Il2rg deletion, since Il2rg is located in the X chromosome.

Conditioning Regimens

Total body irradiation: Prior to sub-lethal irradiation conditioning, the radiation dose was calibrated using an acrylic phantom placed in the same experimental set up to be used for animal irradiation. The measurements were taken using an ion chamber with a solid water phantom build-up. Animals in Phase 1 and Phase 2 were exposed to total body irradiation (Day 1) as follows: Dosimetry measurements were taken with scanned inLight nanodot from Landauer and using a Farmer electrode chamber. The theoretical dose rate, duration of irradiation and the actual time of irradiation for each subset of animals were documented. Body measurements parallel to the radiation beam were taken from 10 selected animals in each phase to calculate the midplane dose rate and radiation exposure times. Chest depth was measured from the sternum to equivalent point in the intrascapular area in millimeter with 2 digits with a caliper. Animals in Phase 1 received a total body irradiation dose of either 140, 160, 180 or 200 cGy (±5% of target dose). The animals in Phase 2 received a total irradiation dose of 200 cGy (±5% of target dose), based on the data obtained in Phase 1. The dose rate of the ⁶⁰Co gamma source (Theratron 1000) was approximately 160 cGy per minute. The optimal irradiation dose for myeloablation was considered to be the highest sublethal dose.

Myeloablation: Busulfan (6 mg/mL) was prepared in 0.9% Sodium Chloride for Injection USP solution and administered via intra-peritoneal injection at a concentration of 15 mg/kg for males and 20 mg/kg for females on Day −2 and Day −1 relative to CD34⁺ hHSPCs administration. The day that hHSPCs were administered was considered as Day 1 for the purposes of this study.

CD34⁺ human hHSPCs (Adult Mobilized)

Adult mobilized CD34⁺ hHSPCs from three different donors at the concentration of 5.0 x 10⁶/mL were obtained cryopreserved in liquid nitrogen vapor phase from HemaCare BioResearch Products (catalog Nos. M34C-GCSF-2 (donor 1 and 2) and M34C-2 (donor 3)). Cell formulations were prepared on the day of dosing, prior to dose administration. The vials of cells were thawed in a water bath at 37°C and resuspended in CellGenix® GMP SCGM Medium/0.1% HSA (thawing media) up to 10 mL. Cells were centrifuged at 300 x g for 5 minutes at room temperature. The cell pellet was resuspended with the PBS 1X pH 7.4/0.1% HSA (injection buffer), counted, and further diluted to achieve ∼4x10⁶ viable cells/mL. Animals were irradiated at 200 cGy or conditioned with busulfan and then received ∼1x10⁶ cells (in a total volume of 0.25 mL) of adult mobilized CD34⁺ hHSPCs. Control groups (non-irradiated, irradiated or busulfan-treated) were administered 0.1% Human Serum Albumin in PBS 1X (Phosphate buffered saline 1X, pH 7.4 without calcium and magnesium and 25% HSA).

Cell counts and viability were performed prior and after dosing. After dosing completion, an aliquot from the remaining volume of the preparation that was drawn up in the syringe was used for cell count and viability. The viability CD34⁺ cells was performed by mixing the cell suspension with acridine orange/propidium iodide solution (AO/PI). The AO/PI allowed the differential staining of live and dead cells. CD34⁺ cell counts were determined with an automated cell counter. Cell count and viability of HL-60 cells was performed by mixing the cell suspension with trypan blue and manual counting with an hemacytometer. Acceptance criteria for viability was considered to be ≥ 70%. The cell formulations were administered once by intravenous slow bolus injection (i.e. 0.25 mL administered over 30-60 seconds) in the tail vein using a 27G needle attached to a syringe on Day 1. Based on unpublished data obtained at the facility, all injections of CD34⁺ hHSPCs were administered within 8 hours of irradiation and 24 ± 2 hours of busulfan, when applicable. The number of cells administered intravenously to each animal was ∼1x10⁶ cells ±20% for test groups irradiated or busulfan-treated. Negative control animals (non-irradiated, irradiated or busulfan-treated) received a single intravenous slow bolus tail vein injection of injection buffer (0.25 mL) with the same conditions as the treated groups. Cell formulations were dosed within 3 hours post-concentration adjustment in the syringe.

Experimental Design

Phase 1 animals were irradiated as shown in Table 1. Following Phase 1, the test and reference items/vehicle were administered to Phase 2 animals via a single intravenous slow bolus injection as shown in Table 2.

Table 1.

Phase 1 – Radiation dose optimization.

Treatment Group	Irradiation dose (cGy)	Irradiation dose rate (cGy/min)	Number of animals
Treatment Group	Irradiation dose (cGy)	Irradiation dose rate (cGy/min)	Male	Female
Group 1	140	∼160	4	4
Group 2	160		4	4
Group 3	180		4	4
Group 4	200		4	4

Table 2.

Phase 2 –Characterization of the NCG for cell therapy assessments.

Treatment Group	Test Item dose (Cells/Mouse)	Test Item dose Conc. (Cells/mL)	Dose Volume, (µL/mouse)	Donor	Conditioning	Number of animals
Treatment Group	Test Item dose (Cells/Mouse)	Test Item dose Conc. (Cells/mL)	Dose Volume, (µL/mouse)	Donor	Conditioning	M	F
Group 5 (PBS control^a)	0	0	250	N/A	None	16	16
Group 6 (PBS controla)	0	0			200 cGy^d	8	8
Group 7 (PBS controla)	0	0			busulfan^b	6	6
Group 8 (CD34⁺ hHSPCs mobilized)	1x10⁶	4x10⁶		1	200 cGy^d	5	5
				2		3	3
				3		3	3
Group 9 (CD34⁺ hHSPCs mobilized)	1x10⁶	4x10⁶		1	busulfan^b	5	5
				2		3	3
				3		3	3
Group 10 (positive control^c)	2x10⁶	8x10⁶		N/A	200 cGy^d	4	4

Animals were administered 0.25 mL of reference item/vehicle (formulation used to resuspend the cells).

Busulfan was administered via intra-peritoneal injection on Day −2 and Day −1 at 15 mg/kg for males and at 20 mg/kg for females.

Animals were administered HL-60 (∼2x10⁶ cells ±20%/mouse). Necropsies were performed on Day 40 post-injection.

The dose rate of irradiation was approximately 160 cGy per minute.

M: male; F: Female; Conc.: Concentration

Supportive Care

Acidified water was given throughout the study to prevent bacterial growth. Sterile water gel packs were provided at least two days before irradiation or busulfan administration, and for the duration of the study.

In-Life Assessments

NCG mice were monitored for clinical signs, mortality, body weights, hematology, clinical chemistry, and flow cytometry.Table 3

Table 3.

Mean Weekly Body Weights in Animals From Phase 2.

	PBS, No Conditioning, No Cells		PBS, 200 cGy		PBS, Busulfan		CD34⁺ hHSPCs Mobilized (200 cGy)		CD34⁺ hHSPCs Mobilized (Busulfan)
Week	M	F	M	F	M	F	M	F	M	F
−1	25.8 ± 1.6	21.6 ± 1.3	26.5 ± 1.1	20.8 ± 1.0	25.5 ± 1.2	20.8 ± 1.2	26.5 ± 1.3	20.6 ± 1.6	26.0 ± 1.2	20.7 ± 0.9
1	25.9 ± 1.6	22.1 ± 1.6	25.8 ± 0.9	20.8 ± 1.3	26.3 ± 0.8	22.0 ± 1.4	25.9 ± 1.7	20.6 ± 1.4	26.2 ± 0.9	21.3 ± 1.3
2	26.4 ± 1.4	22.8 ± 1.4	26.4 ± 1.2	21.3 ± 0.7	25.8 ± 1.0	21.5 ± 0.8	26.2 ± 1.3	21.6 ± 1.7	26.5 ± 1.2	21.8 ± 1.2
3	26.5 ± 1.7	23.1 ± 1.0	26.3 ± 1.3	21.5 ± 0.9	26.3 ± 0.5	21.5 ± 0.8	26.3 ± 1.6	21.5 ± 1.1	26.7 ± 1.0	21.9 ± 1.1
4	27.3 ± 1.7	24.1 ± 1.6	26.8 ± 1.0	22.0 ± 1.3	27.3 ± 0.5	23.0 ± 1.7	26.9 ± 1.5	21.9 ± 1.6	27.5 ± 1.3	22.5 ± 1.0
5	27.9 ± 1.8	24.1 ± 1.6	27.0 ± 1.2	22.9 ± 1.2	28.0 ± 1.3	22.0 ± 1.1	27.5 ± 1.0	22.3 ± 1.4	28.0 ± 1.7	22.7 ± 1.1
6	28.0 ± 2.0	24.2 ± 1.8	27.6 ± 1.1	23.6 ± 0.7	27.8 ± 0.8	22.7 ± 1.2	27.3 ± 1.1	23.3 ± 1.5	27.8 ± 1.5	23.2 ± 1.3
7	28.1 ± 1.7	24.5 ± 1.7	27.9 ± 0.8	23.8 ± 1.0	28.0 ± 0.6	23.2 ± 1.0	27.8 ± 0.8	23.6 ± 1.6	28.2 ± 1.3	23.5 ± 1.4
8	28.4 ± 1.8	24.6 ± 1.6	28.4 ± 1.7	23.3 ± 1.3	28.0 ± 0.6	23.2 ± 0.8	28.4 ± 0.9	23.7 ± 1.3	28.5 ± 1.1	22.9 ± 1.4
9	28.6 ± 1.9	24.6 ± 1.5	28.1 ± 1.6	23.1 ± 1.1	28.2 ± 1.0	23.2 ± 0.8	28.1 ± 1.3	23.2 ± 1.3	28.2 ± 1.7	23.2 ± 2.2
10	28.3 ± 2.0	24.4 ± 1.4	27.9 ± 1.0	22.6 ± 1.2	27.3 ± 1.0	22.7 ± 0.8	27.3 ± 1.7	22.8 ± 1.3	27.6 ± 1.6	22.6 ± 1.7
11	28.8 ± 1.7	25.0 ± 1.6	28.1 ± 1.4	23.9 ± 1.4	27.5 ± 1.0	23.2 ± 1.3	28.3 ± 1.3	23.2 ± 1.4	28.1 ± 1.3	23.3 ± 1.4
12	28.8 ± 1.7	24.9 ± 1.9	28.4 ± 1.3	23.5 ± 0.9	28.0 ± 1.0	23.8 ± 0.8	28.5 ± 1.1	23.8 ± 1.4	28.3 ± 1.2	23.5 ± 1.6
13	29.1 ± 1.9	25.1 ± 1.3	28.4 ± 1.3	24.4 ± 1.3	28.4 ± 1.1	24.0 ± 0.7	28.3 ± 1.6	23.8 ± 1.5	28.7 ± 1.5	23.9 ± 1.6
14	29.4 ± 1.9	25.1 ± 1.5	28.6 ± 1.3	24.6 ± 1.1	28.6 ± 0.9	24.6 ± 1.7	28.9 ± 1.4	24.5 ± 1.4	28.7 ± 1.6	24.2 ± 1.4
15	29.1 ± 2.0	25.4 ± 1.2	28.8 ± 1.7	24.3 ± 1.7	28.6 ± 0.9	24.0 ± 1.2	28.8 ± 1.3	24.8 ± 1.5	28.7 ± 1.8	23.8 ± 1.6
16	29.1 ± 1.9	25.1 ± 1.2	28.4 ± 1.2	24.0 ± 1.3	28.2 ± 1.5	24.2 ± 1.3	28.3 ± 1.8	24.6 ± 1.5	28.1 ± 2.0	23.5 ± 1.6
17	29.4 ± 2.2	25.6 ± 1.2	28.9 ± 1.1	24.3 ± 1.3	29.2 ± 1.3	23.8 ± 1.1	28.8 ± 1.2	25.1 ± 1.9	28.5 ± 2.4	24.0 ± 1.5
18	29.7 ± 2.1	25.4 ± 1.2	29.1 ± 1.2	24.3 ± 2.0	28.8 ± 1.3	23.8 ± 1.1	29.1 ± 1.4	25.1 ± 2.3	28.5 ± 2.0	24.4 ± 1.6
19	29.0 ± 2.3	24.9 ± 0.8	28.3 ± 1.3	24.1 ± 1.1	28.2 ± 1.1	23.6 ± 1.1	28.6 ± 1.2	25.1 ± 1.9	27.9 ± 2.6	23.6 ± 1.9
20	29.4 ± 2.0	25.6 ± 1.2	29.1 ± 1.1	25.5 ± 2.4	28.6 ± 0.9	23.8 ± 1.3	29.9 ± 1.4	25.9 ± 1.8	28.4 ± 2.7	24.1 ± 1.5

M = Males; F = Females

Hematology

Whole blood (125 µL) was collected in K₂EDTA 0.5 mL tubes on Weeks 12 and 19 post-injection and analyzed using the Sysmex hematology analyzer in Low Aspiration Mode on the same day of collection. The following parameters were assessed: hematocrit, hemoglobin, mean corpuscular hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCH), red blood cell count (RBC), red blood cell distribution width (RDW), total white blood cell count (WBC), WBC differential (neutrophils, lymphocytes, monocytes, eosinophils, basophils and large unstained cells; absolute and relative), platelet count and reticulocyte (absolute and relative).

Flow Cytometry

Non-terminal whole blood samples were collected in K₃-EDTA tubes, on Weeks 10 and 17 post-injection from negative control groups (non-irradiated, irradiated or busulfan-treated) and from groups that received CD34⁺ hHSPCs following irradiation or busulfan. The blood samples were kept at room temperature with orbital shaking pending flow cytometry analysis. On Week 20 post-injection, terminal spleen and bone marrow samples were collected from the same groups. Following euthanasia, the spleen was removed, and one-third of the spleen was placed on wet ice or at 2 to 8°C then used for flow cytometry analysis. Femurs were excised, trimmed (muscle removed) and placed on wet ice or at 2 to 8°C pending flow cytometry. The identification of the mouse cell (mCD45⁺hCD45^-) and human cell (mCD45^-hCD45⁺) populations by flow cytometry was performed by direct staining of whole blood samples, as well as spleen and bone marrow cell suspensions with APC mouse anti-human CD45 and PE rat anti-mouse CD45. Fixable Viability Dye eFluor 506® (FVD506, Invitrogen) was also included in the staining panel to exclude dead cells from the analysis.

Splenocytes were isolated from spleen using an initial 100 μm cell strainer at 2 – 8°C conditions, followed by dissociation using a gentleMACS^TM Dissociator. The cell suspension was placed in a GentleMACS C tube with ice cold MACS buffer followed by a 70 μm cell strainer. For bone marrow cell preparation, femurs, with the epiphysis cut, were placed in a perforated 0.5 mL tube in a second 1.5 mL tube with PBS 1X and centrifuged at 10,000 x g until the bone marrow was flushed to the collecting tube. Any clumps were dissociated with a pipette and the cell suspension was then passed through a 70 μm cell strainer, rinsed with ice-cold PBS 1X and filtered again. Cell concentrations in whole blood and absolute cell counts in spleen and bone marrow samples were determined using AccuCheck counting beads (Life Technologies/Invitrogen) with a known stock concentration. For whole blood samples, erythrocytes were lysed (PharmLyse Lysing Buffer, BD Biosciences), then cells were blocked and washed. Cells were then stained with the appropriate antibody mix and viability stain for 30 ± 5 minutes on wet ice protected from light. For spleen and bone marrow samples, erythrocytes from the cell suspension were lysed and cells were counted prior to flow cytometry staining. Spleen and bone marrow cell suspensions were lysed with PharmLyse 1X, blocked with TruStain FcX^TM (anti-mouse CD16/32) and Human TruStain FcX^TM (Fc receptor blocking solution) solutions for 10 minutes on wet ice and then incubated with the appropriate antibody mix and viability stain for 30 minutes on wet ice protected from light. Antibodies used were APC mouse anti-human CD45 and PE rat anti-mouse CD45 (BD Biosciences) diluted in PBS 1X with 2% iFBS. Single events were gated based on their height and area in the forward scatter (FSC) parameter. Then, leukocytes were gated based on granularity (side scatter, SSC) and size (FSC) while dead cells were excluded based on FVD506 positivity. Samples were acquired on FACS Lyric flow cytometer (BD Biosciences).

qPCR for Human Alu Sequences

Quantitative real-time PCR (qPCR) was used to determine the expression of human Alu sequences in whole blood (Weeks 12 and 19 post-injection) and tissues (Week 20 post-injection) from NCG mice. Following genomic DNA extraction (gDNA), qPCR reaction was performed using 500 ng of gDNA per well and ALU expression reported as pg/500 ng of mouse gDNA. The following primers and probes were used: forward primer nsP1: 5′- TGG TGG CTC TCT CCT GTA AT -3′, reverse primer nsP1: 5′- GAT CTC GGC TCA CTG CAA GC -3′ and TaqMan Probe nsP1: 5’-/56-FAM/TGA GGC AGG AGA/ZEN/ATC GCT TGA ACC/3IABKFQ/-3' (Integrated DNA technologies) in a QuanStudio 5 Real-Time PCR system with Quantstudio 5 Design and analysis software. The total fluorescence emitted by the released fluorophore was measured at each cycle. A passive reference dye (ROX dye) was included in the qPCR reaction and provided a stable baseline reference to which samples were normalized to compensate for variations in fluorescence between wells or between experiments. Human peripheral blood mononuclear cells (hPBMCs) were used as a positive control and the number of copies per well in samples was extrapolated for ALU qPCR assays from a standard curve of hPBMC in water containing NCG mouse DNA. The following list of tissues were analyzed: liver, brain, kidney, heart, ovaries, testes, spleen, lung, and bone marrow.

Histopathology

The masses, spleen, femur and bone marrow were fixed and preserved in neutral buffered 10% formalin for at least 16 hours and up to 32 hours then processed into paraffin blocks. Hematoxylin and eosin staining, and microscopic analysis were performed on these tissues.

Statistics

Pairwise comparisons of interest were performed comparing each group with PBS control animals that were irradiated with 200 cGy. For each data set with more than two groups, a one-way analysis of variance (ANOVA) was performed and the residuals were tested for normality using a Shapiro-Wilk test. When the distribution of the residuals was significantly different from a normal one (P ≤ 0.05), the results from the ANOVA were rejected and a second ANOVA was performed using square root transformed data. For data sets with negative values, the absolute lowest observed value was added to the data before the transformation. The normality distribution of the residuals from the second ANOVA was tested using a Shapiro-Wilk test.

When the Shapiro-Wilk test was not significant (P > 0.05) a Levene test was performed on the residuals to assess the homogeneity of the group variances. If differences between group variances were not found to be significant (P > 0.05) then the results from the related ANOVA were retained. When significant differences among the group means are indicated by the ANOVA overall F-test (P ≤ 0.05), a Dunnett test was used to perform the group mean comparisons of interest.

When the Shapiro-Wilk test on the residuals of the second ANOVA was significant or when heterogeneous group variances (P ≤ 0.05) were revealed by the Levene test, ANOVA results were rejected and the groups were compared using a non-parametric Kruskal-Wallis test. When the Kruskal-Wallis test was significant (P ≤ 0.05), a Dunn test was used to perform the pairwise group comparisons of interest.

Each group comparison of interest was conducted via a two-sided test at the 5% significance level and the significant results were reported as either P ≤ 0.001, P ≤ 0.01 or P ≤ 0.05, where p represents the observed probability.

Separately for the 2 phases and on combined sexes, the survival function of each group was estimated using the Kaplan-Meier product-limit method applied on daily intervals.

Results

Phase 1

NCG mice well tolerated all irradiation dose levels, and all animals survived until scheduled euthanasia. For hematology parameters, minimal and non-dose related decreases in mean corpuscular volume were noted in males and females irradiated at ≥ 160 cGy at termination on Day 30. Decreases in mean corpuscular volume likely resulted in minimal and non-dose related increases in red blood cell distribution width in males irradiated at ≥ 180 cGy and females irradiated at 200 cGy when compared to animals irradiated at the lowest irradiation dose at 140 cGy. These changes (decreases in mean corpuscular volume and increases in red blood cell distribution width) were not associated with any changes in red blood cell mass parameters (red blood cell count, hemoglobin and hematocrit), and thus were considered not clinically significant (data not shown).

The pathology evaluation did not reveal any irradiation-related findings or organ weight changes. Although, the thymus was not identified in 12/16 male and in 10/16 female animals, which was considered a consequence of the NCG mice phenotype and unrelated to the irradiation dose. Two males that received 200 cGy had minimally decreased cellularity of the femoral bone marrow hematopoietic compartment, which was considered incomplete regeneration following irradiation induced depletion. There were no residual irradiation effects on the femoral bone marrow at 140, 160 or 180 cGy, indicating that these animals had completely recovered from the irradiation conditioning.

There were no significant body weight changes or clinical observations in Phase 1 and Phase 2 related to irradiation, busulfan conditioning or administration of CD34⁺ hHSPCs.

Phase 2

In light of Phase 1 results, the irradiation dose in Phase 2 for myeloablation conditioning was set as 200 cGy. Following conditioning with irradiation or busulfan-treatment, Figure 1 NCG mice were administered a slow intravenous bolus injection of CD34⁺ hHSPCs, with a pre-injection and post-injection viability above 70%. Details on dose formulation cell viability are presented on Table 4. Irradiation or administration of busulfan only or injection of CD34⁺ hHSPCs to mice myeloablated with either conditioning regimens did not result in significant body weight changes or clinical observations. Animals in the positive control group administrated with HL-60 cells exhibited expected tumor-related clinical signs, such as palpable masses (Table 5 below), decreased activity, hunched back posture, thinness, weakness, closed eyes, erected fur and increased respiratory rate. Clinical observations in this group increased in incidence and severity over the duration of the observation period and had to be humanely euthanized.

Figure 1.

Body weights of Phase 1 and Phase 2 male and female animals. Average body weights from Week −2 to Week 5 for Phase 1 animals that underwent irradiation and Week −2 to Week 20 for Phase 2 animals that were either irradiated with 200 cGy or treated with busulfan and then injected with CD34⁺ hHSPCs. Data points represent means and error bars represent standard deviation.

Table 4.

Dose Formulation Cell Viability.

Conditioning #	Cell Type	Occasion	Mean Live Cell Concentration (x10⁶ Cells/mL)	Nominal Cell concentration (x10⁶ Cells/mL)	Relative error (%)	Mean Viability (%)
200 cGy and busulfan	hCD34+	Post thawing	9.33	—	—	94.9
200 cGy and busulfan		Pre-dose	3.97	4.00	−0.7	93.8
200 cGy		Post-dose	4.08	4.00	2.0	93.9
Busulfan		Post-dose	5.03	4.00	25.8^b	94.2
200 cGy and busulfan	hCD34+	Post thawing	9.06	—	—	94.6
200 cGy and busulfan		Pre-dose	3.88	4.00	−3.0	94.1
200 cGy		Post-dose	4.63	4.00	15.8	94.2
Busulfan		Post-dose	4.75	4.00	18.8	95.1
200 cGy (positive control)	HL-60^a	Post thawing	5.88	—	—	64.0
		Pre-dose	8.00	8.00	0.0	67.0
		Post-dose	NA	8.00	NA	NA
	HL-60	Post thawing	6.57	—	—	73.0
		Pre-dose	7.99	8.00	−0.1	78.0
		Post-dose	9.55	8.00	19.4	75.0
200 cGy and busulfan	hCD34+	Post thawing	7.15	—	—	93.1
200 cGy and busulfan		Pre-dose	4.51	4.00	12.8	93.1
200 cGy		Post-dose	3.97	4.00	−0.7	91.6
Busulfan		Post-dose	4.45	4.00	11.3	91.3

Cells discarded due to low viability;

RE did not meet acceptance criteria; h = Human; NA = Not applicable.

Table 5.

Palpable Masses Observed in Animals Administered HL-60.

Animal ID	Mass Nº	Site	First to Last Seen (Days Post-injection)
10003	1	Abdominal	38-38
10004	1	Dorsal-thoracic	38-40
10503	1	Left flank	35-37
	1	Urogenital	35-37
	1	Urogenital	35-37

Hematology

Irradiation at 200 cGy or administration of busulfan resulted in changes in hematology parameters when compared to non-conditioned animals. Administration of CD34⁺ hHSPCs to mice irradiated or busulfan-treated also induced changes in hematology parameters. These changes, presented in Table 6, occurred in red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCH), red blood cell distribution width (RDW), reticulocyte counts (RETIC), white blood cell counts (WBC), neutrophil counts (NEUT), lymphocyte counts (LYMPH), monocyte counts (MONO), eosinophil counts (EOS), and platelet count (PLT).

Table 6.

Conditioning and mobilized CD34⁺ hHSPCs-related hematology changes.

Treatment	PBS		PBS		PBS		CD34⁺ hHSPCs		CD34⁺ hHSPCs
Cells/Mouse	0	0	0	0	0	0	1x10⁶	1x10⁶	1x10⁶	1x10⁶
Conditioning	None	None	200 cGy	200 cGy	Busulfan	Busulfan	200 cGy	200 cGy	Busulfan	Busulfan
Sex	M	F	M	F	M	F	M	F	M	F
RBC (x10⁶/µL)
Week 12	9.739C NA	9.463 NA	8.990 (0.92x)	8.755 (0.93x)	9.258 (0.95x)	8.808 (0.93x)	7.783 C (0.80x)	7.228 (0.76x)	7.662 C (0.79x)	6.722 D (0.71x)
Week 19	9.505 NA	9.443 NA	9.091 (0.96x)	8.453 (0.89x)	9.082 (0.96x)	8.714 (0.92x)	6.870 (0.72x)	6.630 (0.70x)	6.147 D (0.65x)	5.878 D (0.62x)
HGB (g/dL)
Week 12	15.16C NA	15.08 NA	14.20 (0.94.x)	14.35 (0.95x)	14.38 (0.95x)	14.23 (0.94x)	13.15 C (0.87x)	13.08 (0.87x)	13.34 C (0.88x)	12.22 D (0.81x)
Week 19	14.80 NA	15.01 NA	14.34 (0.97x)	13.78 (0.92x)	13.98 (0.94x)	14.18 (0.94x)	12.23 D (0.83x)	12.35 (0.82x)	11.45 E (0.77x)	11.23 D (0.75x)
HCT (%)
Week 12	52.83 B	51.11 NA	49.65 (0.94x)	49.19 (0.96x)	50.76 (0.96x)	49.00 (0.96x)	45.65 B (0.86x)	44.31 (0.87x)	46.55 A (0.88x)	42.08 D (0.82x)
Week 19	51.76 NA	51.07 NA	50.39 (0.97x)	47.55 (0.93x)	49.10 (0.95x)	48.68 (0.95x)	42.95 D (0.83x)	42.32 (0.83x)	39.98 E (0.77x)	38.68 D (0.76x)
MCV (fL)
Week 12	54.24 NA	54.03 NA	55.24	56.31	54.82	55.83	58.76 (1.08x)	61.50 (1.14x)	60.81 E (1.12x)	63.55 D (1.18x)
Week 19	54.51 NA	54.08 NA	55.45	56.60	54.06	56.24	62.95 (1.15x)	63.97 (1.18x)	65.14 D (1.20x)	65.77 E (1.22x)
MCH (pg)
Week 12	15.57 NA	15.94 NA	15.79	16.44	15.54	16.22	16.93 (1.09x)	18.16 D (1.14x)	17.43 D (1.12x)	18.36 (1.15x)
Week 19	15.59 NA	15.89 NA	15.78	16.40	15.38	16.38	17.95 (1.15x)	18.66 (1.17x)	18.62 D (1.19x)	19.08 E (1.20x*)
RDW (%)
Week 12	19.32 NA	18.84 NA	18.58	18.33	19.24	18.20	19.61	19.04	19.20	19.37 D
Week 19	18.67 NA	18.30 NA	18.34	18.34	18.60	18.10	19.97 A (1.07x)	17.97 (1.03x)	20.13 B (1.08x)	19.65 (1.07x)
RETIC (x10⁹/L)
Week 12	453.40 NA	464.07 NA	418.03 (0.92x)	385.23 (0.83x)	474.10	431.07	367.62 (0.81x)	358.13 (0.77x)	360.75 A (0.80x)	404.37 (0.87x)
Week 19	475.13 NA	444.69 NA	424.14 (0.89x)	376.73 (0.85x)	447.56	435.02	364.84 (0.77x)	311.07 (0.70x)	330.15 B (0.69x)	318.32 (0.72x)
PLT (x10³/µL)
Week 12	1531.8 NA	12.96 NA	1099.1 (0.72x)	1066.3 (0.82x)	1124.4 (0.73x)	1090.5 (0.84x)	1208.6 (0.79x)	938.2 (0.72x)	1163.5 (0.76x)	816.7 A (0.63x)
Week 19	1570.5 NA	1308.7 NA	1339.4 (0.85x)	1195.0	1272.6 (0.81x)	1362.0	1149.4 (0.73x)	1080.5 (0.83x)	1106.7 (0.70x)	997.6 (0.76x)
WBC (x10³/µL)
Week 12	1.242 NA	1.624 NA	1.213	1.069	1.092	1.515	0.911 A (0.73x)	0.878 (0.54x)	0.763 C (0.61x)	0.630 D (0.39x)
Week 19	1.668 NA	1.551 NA	1.098	1.230	0.894	1.304	0.717 (0.42x)	0.921 (0.59x)	0.554 E (0.33x)	0.537 E (0.35x)
NEUT (x10³/µL)
Week 12	0.794 NA	1.031 NA	0.845	0.763	0.782	1.125	0.622 A (0.78x)	0.611 (0.59x)	0.452 C (0.57x)	0.372 E (0.36x)
Week 19	1.167 NA	1.144 NA	0.748	0.901	0.648	1.002	0.459 (0.39x)	0.601 (0.53x)	0.335 F (0.29x)	0.309 E (0.27x)
LYMPH (x10³/µL)
Week 12	0.306 NA	0.441 NA	0.236	0.179	0.200	0.235	0.231	0.184	0.233	0.200
Week 19	0.340B NA	0.232 NA	0.183	0.191	0.128	0.158	0.164 (0.48x)	0.225	0.150 (0.44x)	0.177
MONO (x10³/µL)
Week 12	0.128 NA	0.144 NA	0.126	0.125	0.102	0.148	0.054 (0.42x)	0.081 (0.56x)	0.074 (0.58x)	0.057 (0.40x)
Week 19	0.168 NA	0.161 NA	0.156	0.130	0.110	0.138	0.091 (0.54x)	0.092 (0.57x)	0.065 F (0.39x)	0.048 E (0.30x)
EOS (x10³µL)
Week 12	0.013 D NA	0.008 NA	0.004	0.005	0.008	0.008	0.003	0.003	0.005	0.002
Week 19	0.014 NA	0.013 NA	0.010	0.008	0.004	0.006	0.002 (0.14x)	0.004 (0.31x)	0.002 D (0.14x)	0.003 (0.23x)

M = Males F = Females. NA = Not applicable; - = No clinically relevant changes when compared to non-conditioned group; Numerical values in brackets indicate mean fold change relative to the control non-conditioned group mean.Significant differences when compared to PBS irradiated control group.: A - P ≤ 0.05, B - P ≤ 0.01, C - P ≤ 0.001 (Dunnett) D - P ≤ 0.05, E - P ≤ 0.01, F - P ≤ 0.001 (Dunn)

Conditioning

Administration of busulfan or irradiation at 200 cGy resulted in comparable minimal decreases in red blood cell mass parameters (RBC, HGB, and HCT) and platelets in mice of both sexes at Weeks 12 and 19 post-injection relative to the PBS irradiated control group (Figure 2A, D, and E). Minimal decreases in reticulocyte counts were observed in irradiated mice only at Weeks 12 and 19 post-injection (Figure 2B).

Figure 2.

Hematology parameters in NCG mice conditioned either with irradiation or busulfan-treatment and injected with adult mobilized CD34⁺ hHSPCs. Changes in hematology parameters at Weeks 12 and 19 post-injection of adult mobilized CD34⁺ hHSPCs, as reported in Table 6. P values were calculated with Dunnett test (* = P ≤ 0.05, ** = P ≤ 0.01, and *** = P ≤ 0.001) and Dunn test (# = P ≤ 0.05, ## = P ≤ 0.01, and ### = P ≤ 0.001. Values represent the means of each group and error bars represent standard deviation.

Mobilized CD34⁺ hHSPCs

Administration of CD34⁺ hHSPCs in mice irradiated at 200 cGy or busulfan-treated was associated with minimal to moderate and progressive decreases in red blood cell mass parameters (RBC, HGB, and HCT) and reticulocyte counts from Week 12 to 19 (Figure 2A, B, and E). Decreases in red cell mass and reticulocytes were comparable in magnitude between irradiated or busulfan-treated, while mice injected with CD34⁺ hHSPCs had more marked decreases compared to mice irradiated or busulfan-treated only. These exacerbated decreases in red cell mass and reticulocyte in mice that received mobilized CD34⁺ hHSPCs were noted with concurrent minimal and progressive increases in MCV and MCH in all groups from Week 12 to 19, which resulted in minimally increased RDW in all groups at Week 19 only. In addition, there were minimal to mild and generally progressive decreases in platelets in mice irradiated or busulfan-treated and injected with CD34⁺ hHSPCs at Weeks 12 and 19 (Figure 2D). This change, of comparable magnitude between groups that received CD34⁺ hHSPCs irrespective of their conditioning, exhibited a generally greater magnitude than in mice irradiated or busulfan-treated only. Other CD34⁺ hHSPCs-related changes in hematology parameters included mild to moderate and generally progressive decreases in WBC, neutrophils (Figure 2C), and monocytes in all groups at Weeks 12 and 19, mild decreases in eosinophils in all groups at Week 19 and mild decreases in lymphocytes in male mice irradiated or busulfan-treated at Week 19 (Figure 2F). These changes were attributed to the administration of CD34⁺ hHSPCs as they were not observed in mice irradiated or busulfan-treated only. Positive control animals that received HL-60 cells that survived until scheduled termination only included two males, where in one male, hematology changes consisted of moderately decreased red blood cell mass parameters (RBC, HGB, and HCT) and leucocytes (absolute and differential) on Day 40. These changes were comparable to those observed in other mice from this group that were terminated early.

Flow Cytometry

Whole Blood

Human CD45⁺ cells were observed in whole blood samples from animals at Weeks 10 and 17 post-injection of mobilized CD34⁺ hHSPCs following irradiation at 200 cGy or treatment with busulfan. The concentration of human CD45⁺ cells was comparable between irradiated and busulfan-treated animals at each timepoint. The chimerism ranged from 8.7% to 50.1% at Week 10, and from 2.2% to 75.8% at Week 17 (mean values in Table 7, Figure 3A), with slightly higher percentages in busulfan-treated mice compared to irradiated animals. With this chimerism, the hCD45⁺ cell concentration ranged from 53,033 to 535,854 cells/mL at Week 10, and from 11,764 to 430,163 cells/mL at Week 17, presented as averages for irradiated and busulfan-treated groups in Table 7 and by donor in Figure 3B, C. Importantly, mouse CD45⁺ cell concentration seemed to be lower in animals conditioned with busulfan compared to irradiated animals, which could contribute to the higher chimerism observed in busulfan-treated animals (Table 7, Figure 3D). Differences between CD34⁺ hHSPC donors were observed. Furthermore, for each of the three different CD34⁺ hHSPC donors, there was a tendency to have a higher chimerism in busulfan-treated animals compared to irradiated animals at both timepoints (Figure 3B).

Table 7.

Chimerism and cell concentration or absolute cell counts of hCD45⁺ and mCD45⁺ cell populations in peripheral whole blood, spleen and bone marrow of NCG mice engrafted with adult mobilized CD34⁺ hHSPCs

	Conditioning		Week 10		Week 17		Week 20
	Conditioning		200 cGy	Busulfan	200 cGy	Busulfan	200 cGy	Busulfan
Chimerism	Peripheral blood	M F	23.2% ± 8.8 (N = 20) 21.8% ± 8.7 24.4% ± 9.2	32.7% ± 10.6 (N = 20) 29.7% ± 9.9 35.1% ± 11.0	10.4% ± 5.7 (N = 19) 7.4% ± 4.2 13.1% ± 5.6	22.4% ± 15.4 (N = 21) 14.8% ± 7.1 22.4% ± 15.4	NA	NA
	Spleen	M F	NA	NA	NA	NA	35.0% ± 15.0 (N = 15) 27.0% ± 10.9 42.1% ± 15.1	53.7% ± 12.9 (N = 17) 53.8% ± 13.8 53.7% ± 12.3
	Bone Marrow	M F	NA	NA	NA	NA	35.4% ± 23.5 (N = 19) 30.1% ± 22.4 40.1% ± 24.7	59.9% ± 23.7 (N = 19) 51.6% ± 23.1 69.1% ± 22.0
hCD45⁺ concentration (x10⁶/mL)	Peripheral blood	M F	0.188 ± 0.129 (N = 20) 0.198 ± 0.147 0.180 ± 0.118	0.172 ± 0.071 (N = 20) 0.169 ± 0.077 0.174 ± 0.070	0.061± 0.044 (N = 19) 0.036 ± 0.019 0.083 ± 0.049	0.097 ± 0.087 (N = 21) 0.058 ± 0.024 0.097 ± 0.087	NA	NA
hCD45⁺ absolute cell count (x10⁶)	Spleen	M F	NA	NA	NA	NA	0.426 ± 0.412 (N = 15) 0.411 ± 0.529 0.439 ± 0.315	1.057 ± 0.771 (N = 17) 1.054 ± 0.918 1.064 ± 0.464
hCD45⁺ absolute cell count (x10⁶)	Bone Marrow	M F	NA	NA	NA	NA	3.840 ± 3.779 (N = 19) 2.665 ± 2.519 4.898 ± 4.539	6.717 ± 4.745 (N = 19) 4.828 ± 3.751 8.815 ± 5.042
mCD45⁺ concentration (x10⁶/mL)	Peripheral blood	M F	0.591 ± 0.311 (N = 20) 0.689 ± 0.416 0.510 ± 0.171	0.367 ± 0.170 (N = 20) 0.411 ± 0.214 0.331 ± 0.122	0.533 ± 0.2 (N = 19) 0.494 ± 0.207 0.569 ± 0.197	0.336±0.136 (N = 21) 0.355±0.136 0.336±0.136	NA	NA
mCD45⁺ absolute cell count (x10⁶)	Spleen	M F	NA	NA	NA	NA	0.666 ± 0.564 (N = 15) 0.753 ± 0.711 0.590 ± 0.434	0.785 ± 0.309 (N = 17) 0.694 ± 0.199 0.951 ± 0.417
mCD45⁺ absolute cell count (x10⁶)	Bone Marrow	M F	NA	NA	NA	NA	5.295 ± 2.317 (N = 19) 4.766 ± 1.924 5.770 ± 2.629	3.557 ± 1.645 (N = 19) 3.832 ± 1.826 3.252 ± 1.463

NA = Not applicable; M = Males; F = Females

Figure 3.

Percent chimerism of hCD45⁺ cells and absolute counts of hCD45⁺ and mCD45⁺ cells across different CD34⁺ hHSPC donors in various tissues. Orange datasets represent irradiation while yellow datasets represent busulfan treatment. Male (black) and female (red) data points were only indicated separately when there were significant differences between sexes (A, I). (A) Whole blood percent chimerism of hCD45⁺ cells at 10 and 17 weeks post-injection of CD34⁺ hHSPCs. (B) As in (A) but with separation of CD34⁺ hHSPC donor-specific data and no separation of male and female data points. (C) Whole blood hCD45⁺ cell concentration at 10 and 17 weeks post-injection of CD34⁺ hHSPCs with CD34⁺ hHSPC donor-specific data presented. (D) Whole blood mCD45⁺ cell concentration at 10 and 17 weeks post-injection of CD34⁺ hHSPCs with CD34⁺ hHSPC donor-specific data presented. (E) Spleen percent chimerism of hCD45⁺ cells 20 weeks post-injection of CD34⁺ hHSPCs. (F) As in (E) but with separation of CD34⁺ hHSPC donor-specific data. (G) Spleen absolute cell count of hCD45⁺ cells at 20 weeks post-injection of CD34⁺ hHSPCs with CD34⁺ hHSPC donor-specific data presented. (H) Spleen absolute cell count of mCD45⁺ cells at 20 weeks post-injection of CD34⁺ hHSPCs with CD34⁺ hHSPC donor-specific data presented. (I) Bone marrow percent chimerism of hCD45⁺ cells 20 weeks post-injection of CD34⁺ hHSPCs. (J) As in (I) but with separation of CD34⁺ hHSPC donor-specific data and no separation of male and female data points. (K) Bone marrow absolute cell count of hCD45⁺ cells at 20 weeks post-injection of CD34⁺ hHSPCs with CD34⁺ hHSPC donor-specific data presented. (L) Bone marrow absolute cell count of mCD45⁺ cells at 20 weeks post-injection of CD34⁺ hHSPCs with CD34⁺ hHSPC donor-specific data presented. Error bars represent maximum and minimum values for each group with median represented by the horizontal lines within each box.

Spleen

Human CD45⁺ cells were observed in spleen samples at Week 20 in animals that received CD34⁺ hHSPCs following irradiation or treatment with busulfan. The chimerism (Figure 3E, F), absolute cell count (Figure 3G) and the frequency of this cell population seemed higher in busulfan-treated animals compared to irradiated animals. The chimerism ranged from 13.6% to 60.6% in irradiated animals injected with CD34⁺ hHSPCs, and from 34.9% to 78.3% in busulfan-treated animals injected with CD34⁺ hHSPCs, with an absolute human CD45⁺ cell count ranging from 36.13x10³ to 1,343x10³ cells in irradiated animals, and from 284.2x10³ to 3,360x10³ cells in busulfan-treated animals (mean values presented in Table 7), suggesting a higher engraftment in the busulfan group. However, this difference seemed to be CD34⁺ hHSPC donor dependent. Only two out of the three CD34⁺ hHSPC donors demonstrated a higher hCD45⁺ absolute cell count with busulfan-treatment compared to irradiation, while one CD34⁺ hHSPC donor (Donor 3) demonstrated similar counts in both groups (Figure 3G). The absolute mCD45⁺ cell count was comparable between irradiated and busulfan-treated groups, unlike what was observed in whole blood samples (Figure 3H). No differences were observed between males and females in spleen samples.

Bone Marrow

Human CD45⁺ cells were observed in bone marrow samples at Week 20 in both groups of animals that received CD34⁺ hHSPCs following irradiation or treatment with busulfan. Similar to spleen samples, the chimerism (Figure 3I, J), absolute cell count (Figure 3K) and the frequency in bone marrow for this cell population tended to be slightly higher in the busulfan-treated group compared to the irradiated group, although these were more similar in range.

The chimerism ranged from 4.5% to 88.8% in irradiated animals, and from 8.8% to 91.8% in busulfan-treated animals, with an absolute human CD45⁺ cell count ranging from 91.66x10³ to 15,244x10³ cells in the irradiated group, and from 482.7x10³ to 15,349x10³ cells in the busulfan-treated group. Even when the range of chimerism was similar between the two groups, the mean was higher for busulfan-treated animals at 59.9% compared to the irradiated group at 35.4% (Table 7). The extent of this difference between irradiated and busulfan-treated groups seemed to be driven by CD34⁺ hHSPC Donor 1 and 2 since with Donor 3 (Figure 3J and K), the levels of hCD45⁺ cells and chimerism for busulfan and irradiation conditioning were similar.

Differences between males and females were also observed, particularly in the busulfan-treated group, where the chimerism for females was higher than males (irradiated animals receiving CD34⁺ hHSPCs males: 30.1%, females: 40.1%; busulfan-treated animals receiving CD34⁺ hHSPCs males: 51.6%, females: 69.1%) (Figure 3I).

Similar to whole blood samples, the absolute cell count and frequency of mCD45⁺ seemed lower in busulfan-treated animals compared to the irradiated group (Table 7, Figure 3L), except for one CD34⁺ hHSPC donor (Donor 3). However, slightly higher absolute hCD45⁺ cell counts were present in the busulfan-treated group when compared to the irradiated group. Therefore, it was concluded that the slightly higher chimerism in busulfan-treated animals was not an artefact as in whole blood samples.

Biodistribution (qPCR)

Human Alu sequences of CD34⁺ hHSPCs were detected using qPCR in blood, and tissues (spleen, liver, kidney, bone marrow, brain, heart, lungs, ovaries, and testes) in mice that received CD34⁺ hHSPCs. In all control animals injected with PBS, and subjected to both conditioning regimens, Alu sequences were not detected in any tissues or blood (data not shown). In CD34⁺ hHSPCs injected animals, high levels of Alu sequences were detected in spleen, liver, lungs, and bone marrow, with the highest values obtained from spleen and bone marrow (Table 8, Figure 4). In spleen, irradiated mice had a mean concentration of 129,307.4 ± 80,507.9 pg/500 ng and busulfan-treated mice had a higher mean concentration of 174,878.9 ± 96,633.1 pg/500 ng. Similarly, in bone marrow, irradiated mice had Alu sequence concentrations of 107,143.5 ± 163.775 pg/500 ng and busulfan-treated mice had higher levels at 204,786.2 ± 120,039.3 pg/500 ng (Table 8). Conversely, Alu sequence concentrations were lower in heart (data not shown), kidney (data not shown), brain, liver, testes, ovaries, and blood (Table 7, Figure 4). The lowest concentrations were found in testes with irradiated mice at 68.7 ± 63.7 pg/500 ng and busulfan-treated mice at 284.7 ± 497.9 pg/500 ng.

Table 8.

Concentrations of human Alu sequences detected by qPCR in the blood, spleen, bone marrow, ovaries, and testes.

	Conditioning	Week 20
	Conditioning	200 cGy & CD34⁺ hHSPCs	Busulfan & CD34⁺ hHSPCs
Calculated Quantity (pg/500 ng gDNA)	Blood	133.7 ± 44.4 Males: 73.9/Females 193.5	448.6 ± 428.7 Males: 632.8/Females: 80.1
	Spleen	129,307.4 ± 80,507.9 Males: 103,369.1/Females: 150,058.0	174,878.9 ± 96,633.1 Males: 190,632.9/Females: 157,549.5
	Bone Marrow	107,143.5 ± 163,775.2 Males: 47,377.6/Females: 156,042.9	204,786.2 ± 120,039.3 Males: 159,903.7/Females: 254,156.9
	Ovaries	3643.4 ± 4788.24	1102.50 ± 1601.7
	Testes	68.7 ± 63.7	284.7 ± 497.9

Figure 4.

Biodistribution of human Alu sequences in males and females throughout various tissues. Alu sequence quantification by qPCR in various tissues from males and females either conditioned by irradiation with 200 cGy or treatment with busulfan before injection with CD34⁺ hHSPCs. Values represent means of each group and error bars represent standard deviation.

Pathology Evaluation

Organ Weights

Organ weight changes related to the injection of CD34⁺ hHSPCs with 200 cGy irradiation or busulfan-treatment were present in the spleen. The mean spleen weights (absolute and relative to body and brain) were increased in both male and female animals that received CD34⁺ hHSPCs with busulfan-treatment and in male animals that received CD34⁺ hHSPCs with 200 cGy irradiation (Table 9). These increases were believed to be the result of the engraftment of CD34⁺ hHSPCs within the spleen.

Table 9.

Mobilized CD34⁺ hHSPCs-related irradiation and busulfan-treatment mean absolute and relative organ weight differences compared to irradiated controls.

Sex	Male					Female
Group	PBS	PBS, 200 cGy	PBS, Busulfan	CD34⁺ hHSPCs, 200 cGy	CD34⁺ hHSPCs, Busulfan	PBS	PBS, 200 cGy	PBS, Busulfan	CD34⁺ hHSPCs, 200 cGy	CD34⁺ hHSPCs, Busulfan
Final Body Weight (g)	29.6	29.1	28.6	29.4	28.5	25.7	25.4	23.8	25.7	24.4
Spleen
Absolute	+9.44	—	+32.49	+44.36	+67.68	−32.07*	—	−19.80	+20.00	+39.72
% body	+7.94	—	+35.85	+42.99	+73.43*	−33.08*	—	−15.40	+20.16	+44.33
% brain	+9.50	—	+32.03	+45.20	+68.84*	−34.35*	—	−18.58	+22.16	+41.78

P values were calculated with Dunn test relative to control group (P ≤ 0.05). Values are expressed as percent difference of group means.

Macroscopic Pathology

Macroscopic changes related to the injection of CD34⁺ hHSPCs with 200 cGy or busulfan-treatment were present in the spleen. Three female animals from each group had an enlarged spleen that correlated microscopically with minimal to moderate engrafted round cells.

Microscopic Evaluations

Changes related to the injection of CD34⁺ hHSPCs with 200 cGy or busulfan-treatment were present in the spleen and femoral bone marrow. In the spleen, 10/10 male and 8/11 female animals that received CD34⁺ hHSPCs following irradiation, as well as 11/11 male and 11/11 female animals that received CD34⁺ hHSPCs after busulfan-treatment had minimal to moderate numbers of engrafted round cells in the white pulp, extending into and replacing the hematopoietic tissue. Larger numbers of both male and female animals that received the busulfan-treatment had mild to moderate engraftment than those that received 200 cGy irradiation, which may suggest that busulfan-treatment provided a more favorable environment for engraftment. However, the differences may also be due to normal biological variation. In addition, two females that received CD34⁺ hHSPCs following irradiation, had mild to moderate decreased cellularity of the hematopoietic compartment of spleen, which may reflect a delayed recovery following the irradiation conditioning and depletion and/or possibly replacement by a more dispersed population of CD34⁺ hHSPCs. Occasional female animals that received CD34⁺ hHSPCs and either 200 cGy or busulfan-treatment had minimal increased pigment in the red pulp of the spleen. This likely represented increased turnover of erythrocytes in the spleen secondary to the method of myeloablation. In the bone marrow, 7/10 male and 10/11 female animals that received CD34⁺ hHSPCs with 200 cGy irradiation, as well as 9/11 male and 10/11 female animals that received CD34⁺ hHSPCs with busulfan-treatment had minimal to moderate decreased cellularity of the hematopoietic compartment. In addition, six males from the PBS control group with 200 cGy irradiation and two males from the PBS control group with busulfan-treatment also had minimal to mild decreased cellularity of the hematopoietic compartment of the bone marrow. The higher incidence and severity in groups that received CD34⁺ hHSPCs may be due to the engraftment of these cells in the marrow cavity perhaps slightly slowing the recovery of the hematopoietic tissue following myeloablation. There were no neoplasms associated with the administration of CD34⁺ hHSPCs with either 200 cGy irradiation or with busulfan-treatment. Two males from the positive control group (administered HL-60 cells) had malignant leukemia cells, presumably derived from the HL-60 cells in the bone marrow. Five positive control animals had neoplastic round cells in the femoral bone marrow and/or various other tissues in the body including, the abdominal cavity, kidneys, ovaries, skin and subcutis and/or thoracic cavity. These cells were presumed to be the injected HL-60 cells, and so the neoplasms were diagnosed as malignant leukemia. The observations are presented in Table 10.

Table 10.

Group incidence and severity of mobilized CD34⁺ hHSPCs related irradiation and busulfan-treatment microscopic changes in the spleen and femoral bone marrow.

Sex	Male						Female
Group	PBS	PBS, 200 cGy	PBS, Busulfan	CD34⁺ hHSPCs, 200 cGy	CD34⁺ hHSPCs, Busulfan	HL-60	PBS	PBS, 200 cGy	PBS, Busulfan	CD34⁺ hHSPCs, 200 cGy	CD34⁺ hHSPCs, Busulfan	HL-60
Number of animals	16	8	5	10	11	2	14	8	5	11	11	0
Spleen^a	16	8	5	10	11	2	14	8	5	11	11	0
Engrafted Round Cells	0	0	0	10	11	0	0	0	0	8	11	0
Minimal	0	0	0	3	2	0	0	0	0	2	3	0
Mild	0	0	0	4	5	0	0	0	0	4	5	0
Moderate	0	0	0	2	4	0	0	0	0	2	3	0
Cellularity decreased, hematopoietic	0	0	0	0	0	0	0	0	0	2	0	0
Mild	0	0	0	0	0	0	0	0	0	1	0	0
Moderate	0	0	0	0	0	0	0	0	0	1	0	0
Pigment increased	0	0	0	0	0	0	0	0	0	1	2	0
Minimal	0	0	0	0	0	0	0	0	0	1	2	0
Cellularity increased, hematopoietic	1	0	0	0	0	0	0	0	0	0	0	0
Mild	1	0	0	0	0	0	0	0	0	0	0	0
Femoral bone Marrow^a	16	8	5	10	11	2	14	8	5	11	11	0
Cellularity decreased, hematopoietic	0	6	2	7	9	0	0	0	0	10	10	0
Minimal	0	6	1	4	4	0	0	0	0	4	7	0
Mild	0	0	1	3	4	0	0	0	0	5	3	0
Moderate	0	0	0	0	1	0	0	0	0	1	0	0
Leukemia, malignant	0	0	0	0	0	2	0	0	0	0	0	0

Number examined.

Mortality

Mortality in Phase 2 was limited to one animal from the CD34⁺ hHSPCs group receiving 200 cGy that was euthanized on Day 68 due to severe swelling of the hindlimb. Among the control animals, two females from the PBS control group with no myeloablation, one male and one female from the PBS control with busulfan-treatment group were either euthanized or found dead between Days 77 to 96 post-injection. Three of these animals had spontaneous leukemia/lymphoma in various tissues, which was considered to be the cause of poor body condition and/or death. As expected, 2/4 male and 4/4 female animals in the positive control group (HL-60) were euthanized between Days 27 to 38 post-injection due to poor body condition, which was believed to be due to the presence of neoplastic HL-60 cells in various tissues in 5/6 of these animals. The remaining animal had no microscopic evidence of neoplastic cells in the tissues examined; therefore, the cause of poor body condition was not determined in this animal. Summary of neoplastic findings observed during Phase 2 is presented on Table 11.

Table 11.

Group incidence of neoplastic findings during Phase 2.

Sex	Male						Female
Group	PBS	PBS, 200 cGy	PBS, Busulfan	CD34⁺ hHSPCs, 200 cGy	CD34⁺ hHSPCs, Busulfan	HL-60	PBS	PBS, 200 cGy	PBS, Busulfan	CD34⁺ hHSPCs, 200 cGy	CD34⁺ hHSPCs, Busulfan	HL-60
Number of Animals	16	8	6	11	11	4	16	8	6	11	11	4
Abdominal cavity
Nº of tissues examined	0	0	0	0	0	2	0	0	0	0	0	3
Leukemia/Lymphoma	—	—	—	—	—	2	—	—	—	—	—	3
Bone Marrow
Nº of tissues examined	16	8	6	11	11	4	16	8	6	11	11	4
Leukemia, Malignant	0	0	0	0	0	3	0	0	0	0	0	3
Leukemia/Lymphoma	0	0	1	0	0	0	1	0	0	0	0	0
Heart
Nº of tissues examined	0	0	0	0	0	0	1	0	0	0	0	0
Leukemia/Lymphoma	—	—	—	—	—	—	1	—	—	—	—	—
Kidney
Nº of tissues examined	0	0	0	0	0	0	0	0	0	0	0	1
Leukemia/Lymphoma	—	—	—	—	—	—	—	—	—	—	—	1
Ovaries
Nº of tissues examined	—	—	—	—	—	—	1	0	0	0	0	1
Leukemia/Lymphoma	—	—	—	—	—	—	1	—	—	—	—	1
Skin and subcutis
Nº of tissues examined	0	0	0	0	0	2	0	0	0	0	0	2
Leukemia/Lymphoma	—	—	—	—	—	2	—	—	—	—	—	2
Spleen
Nº of tissues examined	16	8	6	11	11	4	16	8	6	11	11	4
Leukemia/Lymphoma	0	0	1	0	0	0	2	0	0	0	0	0
Thoracic cavity	0	0	0	0	0	0	0	0	0	0	0	1
Nº of tissues examined	—	—	—	—	—	—	—	—	—	—	—	1
Leukemia/Lymphoma

— = Not applicable.

Discussion

In the first phase of this study, the optimized irradiation dose for myeloablation in NCG mice was identified as 200 cGy. This dose corresponds to the historical irradiation dose largely used in preclinical cell therapy research^18,19 and was well tolerated with no mortalities, clinical observations or changes in body weights observed. In the second phase, the NCG mouse model was developed as a test system to assess toxicity, engraftment, biodistribution, and tumorigenicity with different myeloablation regimens: irradiation at 200 cGy or intra-peritoneal busulfan administration.

Pre-Clinical Models for Cell Therapy

Immunodeficient animal models are essential to cell therapy development since mice with intact immune systems will systematically reject human cell engraftments. For a review of humanized mouse models see Shultz et al. 2007 and 2019.^20,21 Currently, there are mouse models that exhibit varying degrees of immunodeficiency. However, the NCG mouse model bearing a NOD background with edits in the genes Prkdc and Il2rg overcomes some of the limitations of previously engineered immunodeficient mouse models such as SCID (severe combined immune deficiency) and NOD (nonobese diabetic) SCID models. For example, while NOD mice have decreased NK cell activity,²² the NK cell activity in NCG mice are absent,²³ thus further decreasing innate immune function and increasing the probability of successful human cell engraftment. This is especially beneficial since NK cell activity is a known deterrent of cell engraftment.^24,25

By having a NOD/Nju background, NCG mice contain a mutation in the gene SIRPα which allows for enhanced engraftment of human hematopoietic stem cells²⁶ while the mutation in the Prkdc gene (protein kinase, DNA-activated, catalytic polypeptide) confers an SCID-like (severe combined immunodeficiency) phenotype which was first characterized in 1983.²⁷ Prkdc is essential for repairing double-stranded DNA breaks, and for generating multiple unique antigen receptors during T and B cell maturation via recombination of V (variable), D (diversity), and J (joining)^28,29 DNA segments. The former function in DNA repair makes these mice sensitive to irradiation while the latter recombination function for mature B and T cell development is reduced in these mice.²⁰ Lastly, the mutation in the Il2rg (interleukin-2-receptor γ-chain) gene results in defective cytokine signaling since the gene product, IL2Rγ, is a necessary component for multiple cytokine receptors including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 leading to additional deficits in innate immunity.^20,21 Notably, mice deficient in Il2rg also have defective NK cell activity.²⁰ In sum, the combination of these genetic modifications makes the NCG mouse model a useful tool for pre-clinical assessment of cell therapy safety and efficacy.

The NSG mouse model is often used for cell therapy toxicology, biodistribution and tumorigenicity assessments.^18,30 Similar to the NSG, the NCG presents defective function for Prkdc and IL2rg genes which renders the strain permissive to human cell engraftment and consequently safety assessment.

Factors Impacting Engraftment

In addition to the mouse model used, other experimental parameters related to animal procedures influence engraftment success to varying degrees including, method of cell injection^31-33 (which also appears to influence risk of graft versus host disease (GvHD) when cord blood is used)^34,35 the interval between myeloablation and cell injection,^36-38 conditioning method,^39,40 conditioning dose,⁴¹ and sex²⁵ and importantly the age of the mice at cell treatment.⁴² Our observation that female mice exhibited increased engraftment rates compared to male mice has been reported in previous studies.²⁵ Indeed, this observation could in part be due to the hemizygous nature of the Il2rg mutation in male mice. When evaluating myeloablation methods, under certain contexts, busulfan-treatment has been shown to be equivalent³⁹ or superior⁴⁰ to irradiation. Furthermore, irradiation may influence subsequent differentiation and lineage fate of the engrafted hHPSCs (see Dorshkind et al. 2020 for review).⁴³

Aside from impacting engraftment success, there are clinically relevant, potential negative side effects associated with irradiation and busulfan-treatment. For example, because irradiation-based conditioning is not cell-specific, especially at high doses, it damages faster replicating cells such as those in gastrointestinal crypts,^44-46 thus increasing the risk of bacterial translocation. To reduce this risk, antibiotics have been used to regulate the gut microflora following irradiation. In addition to preventing infection, antibiotic treatment can reduce GvHD in some experimental contexts. However, antibiotics may also reduce mobilization by impacting endotoxin content in the gut which seems to play a role in mobilization.⁴⁷ Similarly, busulfan which is often used for its myeloablative properties, is known to produce potential adverse effects on vascular endothelial cells.⁴⁸

Engraftment success can also be influenced by experimental parameters associated with the human donors including the type of human cells administered,⁴⁹ source of the cells (for a review comparing peripheral blood or bone marrow stem cell sources see Amouzegar et al. 2019)),⁵⁰ method of collection (venous or arterial access),⁵¹ characteristics of the human donors (including age, sex, body mass index, and genetic background),^52,53 concentration of the cells administered,²⁵ and mobilization strategies.⁵⁴ For example, stem cells from cord blood present a few advantages including permissibility of HLA mismatch and a lower risk for graft versus host disease. However, a major limitation of cord blood is the finite number of HSPCs relative to the amounts found in bone marrow or mobilized peripheral blood. As reviewed by Lund et al. (2015), cord blood has a median time to engraftment longer than 3 weeks, while mobilized peripheral blood stem cells can engraft in 13-15 days.⁴⁹ To overcome these challenges, research has focused on improving ex vivo expansion of cord blood cells to increase their numbers before injection, and on increasing homing of cells to the bone marrow by using intra-bone marrow injections or pathway-mediated approaches through complement, chemokine, and prostaglandin pathways.^49,55

Conversely, when cells are obtained from adults, as mentioned, characteristics including age and sex of the donor impact the success of CD34 cell mobilization response, whereby mobilization has been shown to be less efficient in females^51,52,56 and in older individuals.^51,52 Furthermore, for donor cell mobilization, there is evidence that a combination of G-CSF and plerixafor (Mozobil®) is more effective.⁵⁴ Importantly, these variables do not exist in isolation and can be compounding in nature, for example, busulfan may be metabolized more rapidly in young individuals,⁵⁷ and thus, if using young mice, a higher dose may be considered.⁵⁸ Here, both age and the myeloablation method must be considered. For a review of considerations related to autologous and allogenic hematopoietic cell transplantation, see Duong et al. (2014).⁵⁹

Characterization of the NCG Mice as a Test System for Cell Therapy Assessment

In the present study with NCG mice, we compared irradiation or busulfan-treatment followed by the injection of CD34⁺ hHSPCs from three adult donors then assessed toxicology, engraftment, biodistribution and tumorigenicity.

Conditioning-related clinical pathology changes included minimal decrease in erythrocyte mass and platelets (irradiation or busulfan) and minimal decrease in reticulocytes in irradiated mice. Administration of CD34⁺ hHSPCs in conditioned mice was associated with greater decreases in erythrocyte mass (with secondary increased mean corpuscular volume, mean corpuscular hemoglobin and red blood cell distribution width), reticulocytes and platelets when compared to conditioned mice. Injection of CD34⁺ hHSPC in conditioned mice (irradiation or busulfan-treated) resulted in mildly to moderately decreased total white blood cells, neutrophils, monocytes and eosinophils in all groups while moderately decreased lymphocytes were observed in conditioned male mice.

Engraftment was evaluated by flow cytometry data of hCD45⁺ cell concentration in whole blood at Weeks 10 and 17 post-injection, and absolute hCD45⁺ cell counts in spleen and bone marrow at Week 20. Chimerism obtained with cells collected in adult donors ranged from 2.2% to 75.8% for whole blood, from 13.6% to 78.3% for spleen, and from 4.5% to 91.8% for bone marrow. Interestingly, in whole blood, the level of human cells in females was approximately double that in the males, especially at Week 17. Engraftment was also higher in bone marrow samples of females and differences in chimerism level appeared to be dependent on the CD34⁺ hHSPC donor. Moreover, the chimerism was higher in the busulfan-treated mice compared to irradiated mice, which in whole blood, may be explained in part by the lower level of mCD45⁺ cells in the busulfan-treated group compared to their irradiated counterparts in whole blood samples.

Biodistribution data was assessed by qPCR of human Alu sequences which are ubiquitously spread throughout the human genome⁶⁰ and thus offer a robust method for determining the level of chimerism achieved following CD34⁺ hHSPC injection. We observed Alu sequences across various tissues, in both busulfan-treated and irradiated mice, with highest values in spleen and bone marrow, and lowest in testes.

Spleen organ weights were higher in busulfan-treated mice and irradiated male mice, indicative of round cell engraftment which corroborated with microscopic findings. In bone marrow of conditioned mice injected with CD34⁺ hHSPCs, there was decreased cellularity of the hematopoietic compartment possibly due to delayed recovery and/or the presence of engrafted cells. As expected, neither conditioning group injected with CD34⁺ hHSPCs presented with neoplasms by termination at Week 20 nor did body weights change over the course of the study.

Future Outlook

We observed donor-dependent variation in engraftment as reflected by the varying hCD45⁺ chimerism between CD34⁺ hHSPC donors across tissues. Indeed, donor variability will remain an important consideration in future pre-clinical and clinical trials, and essential to report. Regarding the dose of cells used in the present study (1 x 10⁶ cells/mouse), unlike typical toxicological studies where multiple doses are tested and the pre-clinical doses relate in a predictable manner to the clinical doses, pre-clinical cell therapy dose regimens typically investigate a single dose given limitations with dose translation from murine models to the clinic. It is unclear whether mouse cell doses are even relevant to human doses as the physiological environments and cell signaling cascades are significantly different when studying human cells in a murine model. As an example, mice are lymphocyte predominant amongst the total leukocyte population while humans have a higher population of polymorphonuclear cells.⁶¹ In humans, studies have investigated varying doses of CD34⁺ cells in multiple contexts and patient populations^62-64 with variable results, ultimately demonstrating that the optimal cell dose is heavily dependent on the donor and the stem cell source.⁶²

Rather than informing future dose levels in humans, the pertinence of murine toxicology studies is mostly related to identifying the interaction of gene edited human cells in various organs and confirming that malignancies do not develop over the observation period. Pre-clinical and clinical cell therapy safety assessments will also likely incorporate the typical toxicological paradigm where more significant toxicological effects are accepted for more severe diseases. With autologous CAR-T safety assessments, GvHD is not as much of a concern as with allogenic T cell therapies. However, in both cases, a tissue cross reactivity study is required to assess whether the chimeric TCR reacts with endogenous human tissues. In this assessment, a surrogate that carries the TCR (T cell receptor) binding area, such as a mouse monoclonal antibody, needs to be generated, then applied to healthy human tissues for assessing immunoreactivity. For indications in regenerative medicine, tumorigenicity is an important safety assessment to conduct.⁶⁵ However, the time frame to assess tumorgenicity with confidence remains limited since lymphomas are the most common tumors in mice, and risk increases with age.^66,67 Therefore, when assessing tumorgenicity for extended periods of time post-engraftment, this factor must be considered. Although NSG and NCG mice have some resistance to developing lymphomas when compared to other immunodeficient mouse strains (NOD SCID mice), lymphomas still can occur,⁶⁸ thus, appropriate controls are necessary to help differentiate gene editing related malignancies from spontaneous occurrences.

In future studies, it would be of great interest to precisely characterize histological endpoints in NCG mice. Notably, histopathological signs of GvHD appear to precede clinical signs, pointing towards an increased sensitivity for histopathology to detect GvHD in NSG engrafted mice.⁶⁹ Additionally, it will be relevant to explore cell and gene therapy in unison with the concurrent advancement of gene therapy and the improvement of viral vectors such as lentiviral (LV) and adeno-associated viral (AAV) vectors. LVs insert into DNA potentially causing insertional mutagenesis and increasing the risk of tumors, while AAVs have much lower incidence of integration into DNA, which was further diminished by the generation of recombinant AAVs.⁷⁰ As a result, AAVs are regarded as a safer option for gene therapy approaches with reduced risk of tumorgenicity, in addition to low pathogenicity, and low immunogenicity.⁷⁰ However, depending on whether the target cell is mitotically active, integrating vectors would be necessary for sustained gene expression as non-integrating vectors are preferrable for non-dividing cell targets, therefore LVs are useful for ex vivo gene transfer into hematopoietic and other stem cells, while AAVs are useful for in vivo gene therapy in post-mitotic cells.²⁶ Future studies will likely focus on long-term effects of LV and AAV administration to confidently rule out any potential genotoxicity caused by vector genome integration.^71,72 Whether LV and AAV integration can solely cause increased tumorgenicity remains to be seen, especially since conditioning methods such as busulfan and irradiation, as well as individual comorbidities found in patients can also lead to increased risk of certain cancer types.⁷¹ However, both pre-clinical assessments of tumorgenicity and long-term follow-up clinical studies will be helpful for determining the risk/benefit ratio of these cell and gene therapy strategies.^73-75

Conclusion

The irradiation dose for cell myeloablation in NCG mice targeting the highest sublethal dose which was determined as 200 cGy in Phase 1. The engraftment, biodistribution, and tumorigenicity of mobilized adult CD34⁺ hHSPCs following a single intravenous slow bolus injection into pre-conditioned irradiated or busulfan-treated NCG mice was determined in Phase 2. Human CD34⁺ cells were observed in whole blood, spleen and bone marrow samples in groups injected with mobilized adult CD34⁺ hHSPCs, displaying a chimerism ranging between 2.2% and 91.8%. Biodistribution showed the presence of human Alu sequences at Week 20 across various tissues with the highest levels in the spleen and bone marrow and the lowest in the testes while no Alu sequences were identified in non-injected animals. As expected, there was no neoplasm associated with the administration of mobilized adult CD34⁺ hHSPCs with either 200 cGy irradiation or with busulfan-treatment. Overall, this work illustrates key considerations when designing and interpreting toxicology studies for cell therapies with murine test systems.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Camila Pará

Morgan K. Foret

Simon Authier

References

Weber

Maus

Mackall

. The Emerging Landscape of Immune Cell Therapies. Cel. 2020;181(1):46-62. doi:10.1016/j.cell.2020.03.001.

MacKay

Afshinnekoo

Rub

, et al. The therapeutic landscape for cells engineered with chimeric antigen receptors. Nat Biotechnol. 2020;38(2):233-244. doi:10.1038/s41587-019-0329-2.

Hubbard-Lucey

Tang

. Immuno-oncology drug development goes global. Nat Rev Drug Discov. 2019;18(12):899-900. doi:10.1038/d41573-019-00167-9.

Lin

Lee

Scott

Lin

. Graphical Analyses in the Regulatory Evaluation of Gene Therapy Applications. Ther Innov Regul Sci. 2021;55(2):346-359. doi:10.1007/s43441-020-00219-y.

Peter Marks

, Statement from FDA Commissioner Scott Gottlieb; 2019. , Director of the Center for Biologics Evaluation and Research on new policies to advance development of safe and effective cell and gene therapies https://www.fda.gov/news-events/press-announcements/statement-fda-commissioner-scott-gottlieb-md-and-peter-marks-md-phd-director-center-biologics

Olson

Lin

Liu

Patnaik

. Mouse Models for Cancer Immunotherapy Research. Cancer Discov. 2018;8(11):1358-1365. doi:10.1158/2159-8290.CD-18-0044.

Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER). S9 Nonclinical Evaluation for Anticancer Pharmaceuticals. 2010: Guidance for Industry. https://www.fda.gov/media/73161/download

Kimbrel

Lanza

. Next-generation stem cells - ushering in a new era of cell-based therapies. Nat Rev Drug Discov. 2020;19(7):463-479. doi:10.1038/s41573-020-0064-x.

Pearson

Greiner

Shultz

. Creation of "Humanized" Mice to Study Human Immunity. Curr Protoc Im. 2008;81(1):15-21.

10.

Omer-Javed

Pedrazzani

Albano

, et al. Mobilization-based chemotherapy-free engraftment of gene-edited human hematopoietic stem cells. Cell. 2022;185(13):2248-2264.

11.

Radtke

Humbert

Kiem

. Mouse models in hematopoietic stem cell gene therapy and genome editing. Biochem Pharmacol. 2020;174:113692.

12.

Eberle

Rowe

Fiore

, et al. NCG/CRL as a novel (Charles River) humanized mouse model for pre-clinical oncology studies: immunophenotypic characterization and performance monitoring. Journal for Immunotherapy of Cancer. Online ahead of print. 2019.

13.

Eberle

Rowe

Fiore

Mihalek

Festin

. Enhanced immune responses in human breast and colon cancer following checkpoint therapy in a CD34+ stem cell humanized NCG (HuCD34NCG) mouse model. Journal for ImmunoTherapy of Cancer. 2020;8(suppl 3):A117-A117. doi:10.1136/jitc-2020-SITC2020.0199.

14.

Tarpinian

Rowe

Husanov

, et al. 247 Assessment of sensitivity to a PD-1 check point inhibitor and cisplatin in bladder cancer patient-derived xenografts with various levels of PD-L1 expression in HuCD34NCG mice. Journal for ImmunoTherapy of Cancer. 2020;8(suppl 3):A147-A147. doi:10.1136/jitc-2020-SITC2020.0247.

15.

Rowe

Eberle

Reap

, et al. Abstract 5625: Evaluation of in vivo anti-tumor response of solid tumors in a novel immune cell-humanized NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl mouse model. Cancer Res. 2020;80(16_Supplement):5625-5625. doi:10.1158/1538-7445.am2020-5625.

16.

Bredeson

LeRademacher

Kato

, et al. Prospective cohort study comparing intravenous busulfan to total body irradiation in hematopoietic cell transplantation. Blood. 2013;122(24):3871-3878. doi:10.1182/blood-2013-08-519009.

17.

Petit

Szyper-Kravitz

Nagler

, et al. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol. Jul. 2002;3(7):687-694. doi:10.1038/ni813.

18.

Shultz

Lyons

Burzenski

, et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol. 2005;174(10):6477-6489. doi:10.4049/jimmunol.174.10.6477.

19.

Ghandhi

Smilenov

Shuryak

Pujol-Canadell

Amundson

. Discordant gene responses to radiation in humans and mice and the role of hematopoietically humanized mice in the search for radiation biomarkers. Sci Rep. 2019;9(1):19434. doi:10.1038/s41598-019-55982-2.

20.

Shultz

Ishikawa

Greiner

. Humanized mice in translational biomedical research. Nat Rev Immunol. 2007;7(2):118-130. doi:10.1038/nri2017.

21.

Shultz

Keck

Burzenski

, et al. Humanized mouse models of immunological diseases and precision medicine. Mamm Genome. 2019;30(5-6):123-142. doi:10.1007/s00335-019-09796-2.

22.

Kataoka

Satoh

Fujiya

, et al. Immunologic aspects of the nonobese diabetic (NOD) mouse. Abnormalities of cellular immunity. Diabetes. Mar. 1983;32(3):247-253. doi:10.2337/diab.32.3.247.

23.

Inc. CRL . Immunodeficient Models Data Sheet. 2022. https://www.criver.com/sites/default/files/resources/ImmunodeficientModelsDatasheet%E2%80%94NorthAmerica.pdf

24.

Christianson

Greiner

Schweitzer

, et al. Role of natural killer cells on engraftment of human lymphoid cells and on metastasis of human T-lymphoblastoid leukemia cells in C57BL/6J-scid mice and in C57BL/6J-scid bg mice. Cell Immunol. 1996;171(2):186-199. doi:10.1006/cimm.1996.0193.

25.

McDermott

Eppert

Lechman

Doedens

Dick

. Comparison of human cord blood engraftment between immunocompromised mouse strains. Blood. 2010;116(2):193-200. doi:10.1182/blood-2010-02-271841.

26.

Anguela

High

. Entering the Modern Era of Gene Therapy. Annu Rev Med. 2019;70(1):273-288. doi:10.1146/annurev-med-012017-043332.

27.

Bosma

Custer

Bosma

. A severe combined immunodeficiency mutation in the mouse. Nature. 1983;301(5900):527-530. doi:10.1038/301527a0.

28.

Lewis

. The Mechanism of V(D)J Joining: Lessons from molecular, immunological, and comparative analyses. In: Dixon

, ed. Adv Immunol. Academic Press; 1994:27-150.

29.

Lieber

Pannicke

Schwarz

. The mechanism of vertebrate nonhomologous DNA end joining and its role in V(D)J recombination. DNA Repair (Amst). DNA Repair. 2004;3(8-9):817-826. doi:10.1016/j.dnarep.2004.03.015.

30.

Ishikawa

Yasukawa

Lyons

, et al. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice. Blood. 2005;106(5):1565-1573. doi:10.1182/blood-2005-02-0516.

31.

McKenzie

Gan

Doedens

Dick

. Human short-term repopulating stem cells are efficiently detected following intrafemoral transplantation into NOD/SCID recipients depleted of CD122+ cells. Blood. 2005;106(4):1259-1261. doi:10.1182/blood-2005-03-1081.

32.

van Os

Ausema

Dontje

van Riezen

van Dam

de Haan

. Engraftment of syngeneic bone marrow is not more efficient after intrafemoral transplantation than after traditional intravenous administration. Exp Hematol. Nov. 2010;38(11):1115-1123. doi:10.1016/j.exphem.2010.07.003.

33.

Castello

Podesta

Menditto

, et al. Intra-bone marrow injection of bone marrow and cord blood cells: an alternative way of transplantation associated with a higher seeding efficiency. Exp Hematol. 2004;32(8):782-787. doi:10.1016/j.exphem.2004.05.026.

34.

Frassoni

Varaldo

Gualandi

, et al. The intra-bone marrow injection of cord blood cells extends the possibility of transplantation to the majority of patients with malignant hematopoietic diseases. Best Pract Res Clin Haematol. 2010;23(2):237-244. doi:10.1016/j.beha.2010.06.003.

35.

Rocha

Labopin

Ruggeri

, et al. Unrelated cord blood transplantation: outcomes after single-unit intrabone injection compared with double-unit intravenous injection in patients with hematological malignancies. Transplantation. 2013;95(10):1284-1291. doi:10.1097/TP.0b013e318288ca4d.

36.

Unsgaard

. Optimal Time for Marrow Injection in Mice after Total Body Irradiation. Acta Radiol. 1961;56(4):296. doi:10.3109/00016926109172824.

37.

Weiss

Bullorsky

Ashkenazi

Slavin

. Optimal time interval between myeloablative whole body irradiation and reconstitution with syngeneic bone marrow graft. Bone Marrow Transplant. 1988;3(3):207-210.

38.

Chevaleyre

Duchez

Rodriguez

, et al. Busulfan administration flexibility increases the applicability of scid repopulating cell assay in NSG mouse model. PLoS One. 2013;8(9):e74361. doi:10.1371/journal.pone.0074361.

39.

Choi

Chun

Kim

, et al. Human B cell development and antibody production in humanized NOD/SCID/IL-2Rgamma(null) (NSG) mice conditioned by busulfan. J Clin Immunol. 2011;31(2):253-264. doi:10.1007/s10875-010-9478-2.

40.

Wilkinson

Sergijenko

Langford-Smith

Malinowska

Wynn

Bigger

. Busulfan conditioning enhances engraftment of hematopoietic donor-derived cells in the brain compared with irradiation. Mol Ther. 2013;21(4):868-876. doi:10.1038/mt.2013.29.

41.

Garcia-Perez

van Roon

Schilham

Lankester

Pike-Overzet

Staal

FJT

. Combining Mobilizing Agents with Busulfan to Reduce Chemotherapy-Based Conditioning for Hematopoietic Stem Cell Transplantation. Cells. 2021;10(5)doi:10.3390/cells10051077.

42.

Knibbe-Hollinger

Fields

Chaudoin

, et al. Influence of age, irradiation and humanization on NSG mouse phenotypes. Biol Open. 2015;4(10):1243-1252. doi:10.1242/bio.013201.

43.

Dorshkind

Hofer

Montecino-Rodriguez

Pioli

Rodewald

. Do haematopoietic stem cells age? Nat Rev Immunol. 2020;20(3):196-202. doi:10.1038/s41577-019-0236-2.

44.

Duran-Struuck

Dysko

. Principles of bone marrow transplantation (BMT): providing optimal veterinary and husbandry care to irradiated mice in BMT studies. J Am Assoc Lab Anim Sci. 2009;48(1):11-22.

45.

Yen

Wright

. The gastrointestinal tract stem cell niche. Stem Cell Rev. 2006;2(3):203-212. doi:10.1007/s12015-006-0048-1.

46.

Zhu

Liang

Zhu

, et al.. The effects of myeloablative or non-myeloablative total body irradiations on intestinal tract in mice. Biosci Rep. 2021;26(3):41. doi:10.1042/BSR20202993.

47.

Velders

van Os

Hagoort

, et al. Reduced stem cell mobilization in mice receiving antibiotic modulation of the intestinal flora: involvement of endotoxins as cofactors in mobilization. Blood. 2004;103(1):340-346. doi:10.1182/blood-2002-07-2270.

48.

Zeng

Jia

Yan

Ding

. Vascular endothelium changes after conditioning in hematopoietic stem cell transplantation: role of cyclophosphamide and busulfan. Transplant Proc. 2010;42(7):2720-2724. doi:10.1016/j.transproceed.2010.04.024.

49.

Lund

Boitano

Delaney

Shpall

Wagner

. Advances in umbilical cord blood manipulation-from niche to bedside. Nat Rev Clin Oncol. 2015;12(3):163-174. doi:10.1038/nrclinonc.2014.215.

50.

Amouzegar

Dey

Spitzer

. Peripheral blood or bone marrow stem cells? practical considerations in hematopoietic stem cell transplantation. Transfus Med Rev. 2019;33(1):43-50. doi:10.1016/j.tmrv.2018.11.003.

51.

Wang

Wen

Chen

, et al. Factors associated with peripheral blood stem cell yield in volunteer donors mobilized with granulocyte colony-stimulating factors: the impact of donor characteristics and procedural settings. Biol Blood Marrow Transplant 2008;14(11):1305-1311. doi:10.1016/j.bbmt.2008.09.002.

52.

Vasu

Leitman

Tisdale

, et al. Donor demographic and laboratory predictors of allogeneic peripheral blood stem cell mobilization in an ethnically diverse population. Blood. 2008;112(5):2092-2100. doi:10.1182/blood-2008-03-143677.

53.

Pang

Price

Sahoo

, et al. Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age. Proc Natl Acad Sci U S A. 2011;108(50):20012-20017. doi:10.1073/pnas.1116110108.

54.

DiPersio

Stadtmauer

Nademanee

, et al. Plerixafor and G-CSF versus placebo and G-CSF to mobilize hematopoietic stem cells for autologous stem cell transplantation in patients with multiple myeloma. Blood. 2009;113(23):5720-5726. doi:10.1182/blood-2008-08-174946.

55.

Berglund

Magalhaes

Gaballa

Vanherberghen

Uhlin

. Advances in umbilical cord blood cell therapy: the present and the future. Expert Opin Biol Ther. 2017;17(6):691-699. doi:10.1080/14712598.2017.1316713.

56.

Ings

Balsa

Leverett

Mackinnon

Linch

Watts

. Peripheral blood stem cell yield in 400 normal donors mobilised with granulocyte colony-stimulating factor (G-CSF): impact of age, sex, donor weight and type of G-CSF used. Br J Haematol. 2006;134(5):517-525. doi:10.1111/j.1365-2141.2006.06223.x.

57.

Nguyen

Fuller

Lennon

Leger

Puozzo

CIV

. busulfan in pediatrics: a novel dosing to improve safety/efficacy for hematopoietic progenitor cell transplantation recipients. Bone Marrow Transplant. 2004;33(10):979-987. doi:10.1038/sj.bmt.1704446.

58.

Montecino-Rodriguez

Dorshkind

. Use of busulfan to condition mice for bone marrow transplantation. STAR Protoc. 2020;1(3):100159. doi:10.1016/j.xpro.2020.100159.

59.

Duong

Savani

Copelan

, et al. Peripheral blood progenitor cell mobilization for autologous and allogeneic hematopoietic cell transplantation: guidelines from the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 2014;20(9):1262-1273. doi:10.1016/j.bbmt.2014.05.003.

60.

Deininger

. Alu elements: know the SINEs. Genome Biol. 2011;12(12):236. doi:10.1186/gb-2011-12-12-236.

61.

Mestas

Hughes

. Of mice and not men: differences between mouse and human immunology. J Immunol. 2004;172(5):2731-2738. doi:10.4049/jimmunol.172.5.2731.

62.

Heimfeld

. HLA-identical stem cell transplantation: is there an optimal CD34 cell dose? Bone Marrow Transplant. 2003;31(10):839-845. doi:10.1038/sj.bmt.1704019.

63.

Kalwak

Porwolik

Mielcarek

, et al. Higher CD34(+) and CD3(+) cell doses in the graft promote long-term survival, and have no impact on the incidence of severe acute or chronic graft-versus-host disease after in vivo T cell-depleted unrelated donor hematopoietic stem cell transplantation in children. Biol Blood Marrow Transplant. 2010;16(10):1388-1401. doi:10.1016/j.bbmt.2010.04.001.

64.

Salas

Atenafu

Bautista

, et al. Impact of CD34+ cell dose on reduced intensity conditioning regimen haploidentical hematopoietic stem cell transplantation. Eur J Haematol. 2020;104(1):36-45. doi:10.1111/ejh.13332.

65.

Sato

Bando

Di Piazza

, et al. Tumorigenicity assessment of cell therapy products: The need for global consensus and points to consider. Cytotherapy. 2019;21(11):1095-1111. doi:10.1016/j.jcyt.2019.10.001.

66.

Ward

. Lymphomas and leukemias in mice. Exp Toxicol Pathol. 2006;57(5-6):377-381. doi:10.1016/j.etp.2006.01.007.

67.

Chanut

FJA

Sanvito

Ferrari

, et al. Conditioning Regimens in Long-Term Pre-Clinical Studies to Support Development of Ex Vivo Gene Therapy: Review of Nonproliferative and Proliferative Changes. Hum Gene Ther. 2021;32(1-2):66-76. doi:10.1089/hum.2020.135.

68.

Tillman

Janke

Funk

Vogel

Rehg

. Morphologic and Immunohistochemical Characterization of Spontaneous Lymphoma/Leukemia in NSG Mice. Vet Pathol. Jan. 2020;57(1):160-171. doi:10.1177/0300985819882631.

69.

Blumich

Zdimerova

Munz

Kipar

Pellegrini

. Human CD34(+) Hematopoietic Stem Cell-Engrafted NSG Mice: Morphological and Immunophenotypic Features. Vet Pathol. 2021;58(1):161-180. doi:10.1177/0300985820948822.

70.

Wang

Tai

PWL

Gao

. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019;18(5):358-378. doi:10.1038/s41573-019-0012-9.

71.

Leonard

Tisdale

. A pause in gene therapy: Reflecting on the unique challenges of sickle cell disease. Mol Ther. 2021;29(4):1355-1356. doi:10.1016/j.ymthe.2021.03.010.

72.

Dave

Cornetta

. AAV Joins the Rank of Genotoxic Vectors. Mol Ther. 2021;29(2):418-419. doi:10.1016/j.ymthe.2021.01.007.

73.

Thomas

Ehrhardt

Kay

. Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet. 2003;4(5):346-358. doi:10.1038/nrg1066.

74.

White

Whittaker

Gandara

Stoll

. A Guide to Approaching Regulatory Considerations for Lentiviral-Mediated Gene Therapies. Hum Gene Ther Methods. Aug. 2017;28(4):163-176. doi:10.1089/hgtb.2017.096.

75.

U.S. Department of Health and Human Services, Food and Drug Administration Center for Biologics Evaluation and Research, January 2020. Long term follow-up after administration of human gene therapy products Guidance for Industry 2022. https://www.fda.gov/media/113768/download

Non-Clinical Cell Therapy Development Using the NCG Mouse Model as a Test System

Abstract

Keywords

Introduction

Methods

Animals

Conditioning Regimens

CD34+ human hHSPCs (Adult Mobilized)

Experimental Design

Supportive Care

In-Life Assessments

Hematology

Flow Cytometry

qPCR for Human Alu Sequences

Histopathology

Statistics

Results

Phase 1

Phase 2

Hematology

Conditioning

Mobilized CD34+ hHSPCs

Flow Cytometry

Whole Blood

Spleen

Bone Marrow

Biodistribution (qPCR)

Pathology Evaluation

Organ Weights

Macroscopic Pathology

Microscopic Evaluations

Mortality

Discussion

Pre-Clinical Models for Cell Therapy

Factors Impacting Engraftment

Characterization of the NCG Mice as a Test System for Cell Therapy Assessment

Future Outlook

Conclusion

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iDs

References

CD34⁺ human hHSPCs (Adult Mobilized)

Mobilized CD34⁺ hHSPCs