Fig 1.
Vector copy number (VCN) analysis after GbGM lentiviral (LV) vector and SFFV γ-retro viral (RV) vector gene transduction and transplantation in primary wild-type BoyJ mice.
(A) Portion of vector transduced mouse hematopoietic stem and progenitor cells (LSK) from C57BL/6 mice (CD45.2) were expanded in culture for 14–16 days to measure the VCN in the input injected LSK cells. Mock transduced cells (no vector) were used as negative control whereas SFFV RV transduced cells are used as positive control (B-E) Transduced LSK cells were transplanted into sub-lethally irradiated BoyJ recipient mice. The VCN in (B) PB, (C) BM, (D) spleen, and (E) thymus (analyzed via qPCR) at 8 months after transplant in primary BoyJ recipients are shown. Mean ± standard error of the mean (SEM) is shown for each bar. Each symbol in the bar graph represents an individual animal. Statistics: One-way ANOVA; not significant (ns), *P ≤ .05; **P ≤ .01; ***P ≤ .001, ****P ≤ .0001.
Fig 2.
Analysis of engraftment and cell differentiation of the Mock, GbGM LV and SFFV RV transduced cells in the BM of primary recipient BoyJ mice.
Isolated hematopoietic stem and progenitor (LSK) cells were transduced using GbGM LV, SFFV RV and Mock (no vector) and transplanted into primary recipient BoyJ mice. At 8 months after transplant, bone marrow (BM) cells from the mice were collected and analyzed for hematological reconstitution by flow cytometry. (A) Donor (CD45.2+) cell engraftment in Mock, GbGM LV, and SFFV RV mice. (B-F) Percentages of myeloid and lymphoid (B and T cell) progeny were evaluated by using antibodies against CD11b, B220, CD3, CD4, and CD8 in Mock, GbGM LV, and SFFV RV mice. Each symbol represents an individual animal. Statistics: One-way ANOVA; not significant (ns), *P ≤ .05; **P ≤ .01; ***P ≤ .001, ****P ≤ .0001.
Table 1.
Transplant details and engraftment after secondary transplantation.
Fig 3.
Vector copy number (VCN) analysis after GbGM LV and SFFV RV animals in secondary transplanted BoyJ mice.
Isolated bone marrow cells from the primary recipient BoyJ mice were processed and transplanted into sub-lethally irradiated secondary recipient BoyJ mice to assess the HSC engraftment. The vector copy number (VCN) in (A) peripheral blood (PB), (B) bone marrow (BM), (C) spleen, and (D) thymus was analyzed via qPCR at 10 months after transplant in secondary recipients is shown. Mean and standard error of the mean are shown for each bar. Each symbol in the bar graph represents an individual animal. Statistics: One-way ANOVA; not significant (ns), *P ≤ .05; **P ≤ .01; ***P ≤ .001, ****P ≤ .0001.
Fig 4.
Analysis of engraftment and cell differentiation of the Mock, GbGM LV and SFFV RV transduced cells in the BM of Secondary BoyJ recipient mice.
Isolated BM cells from primary mice were processed and transplanted in secondary recipient BoyJ mice. (A) Donor mouse (CD45.2+) cell engraftment in Mock, GbGM, and SFFV RV secondary mice 10 months after transplant. (B-F) Percentages of myeloid and lymphoid (B and T) cells were evaluated by using antibodies against CD11b, B220, CD3, CD4, and CD8 in Mock, GbGM, and SFFV mice. Each symbol represents an individual animal. Statistics: One-way ANOVA. not significant (ns), *P ≤ .05; **P ≤ .01; ***P ≤ .001, ****P ≤ .0001.
Table 2.
Animals with lymphoproliferation or malignancy.
Table 3.
Animals with donor-derived and host-derived malignancies.
Fig 5.
Liver histopathology and in situ hybridization revealing the origin of lymphoma.
Liver sample from mouse #6 in the GbGM mice group showed a demarcated lymphoma tumor and normal tissue was obtained (H&E stain). The in situ hybridization analysis using the gamma globin vector showed absence of vector sequence in the tumor/lymphoma tissue. However, the in situ analysis showed the presence of vector in hematopoietic cells in the blood vessel and in the liver sinusoidal vessels distributed throughout the liver (100x). The scrambled probe did not show any abnormal staining in the liver tissue.