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Mosaicism was observed in eGFP gene expression in several colonies (Figure
3). Indeed, eGFP was detected in 56% ± 4% of colonies, whereas only 26% ± 5%
of individual cells were eGFP-positive. These results suggest that, on average, only
47% of cells from a single colony contained the SIV vector.
Figure 3.
Fluorescence microscopy after myeloid differentiation of CFC (×100). Freshly isolated CD34
+
cells
were transduced or not with the lentiviral vector (24 hours of culture with lentiviral vector at MOI = 100).
Cells were then cultured for 14 days in the presence of cytokines, to allow myeloid differentiation of transduced
(A) and not transduced (B) CD34
+
cells. Abbreviations: CFU-GEMM, Colony-Forming Unit-Granulocytes,
Erythroid, Macrophage, Megakaryocyte; BFU-E, Burst-Forming Unit-Erythroid; CFU-GM, Colony-Forming
Unit-Granulocytes, Macrophage; CFU-G, Colony-Forming Unit-Granulocytes; CFU-M, Colony-Forming
Unit-Macrophage.
Transplantation of Autologous BM CD34
+
cells transduced
by SIV-Based Vector into Cynomolgus Macaques
We explored the capacity of autologous CD34
+
BM cells transduced ex vivo with
a lentiviral vector to engraft efficiently into macaques after total body irradia-
tion (TBI) with a gamma source at the sublethal dose of 6 Gy. Three groups of
4 animals were used: 1) In Group 1, macaque CD34
+
BM cells (96% ± 1% pure
on average) were obtained from the two humeri before gamma irradiation (Table
1). These cells were cocultured, as described above, with pGASE, which is an
improved version of pRMES8. Indeed, a mean transduction efficiency of 72% ±
4% was obtained (n = 4) at 24 hours and 37% ± 10% of CFC produced eGFP.
Two days after gamma irradiation, 1.4 × 10
6
to 2.9 × 10
6
CD34
+
cells per kg were
injected into both humeri of macaques (Table 1); 2) Group 2 included irradiated
(6 Gy) macaques that did not undergo cell transplantation: 3) Group 3 included
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