Reconstitution of the Myeloid and Lymphoid Compartments after the Transplantation of Autologous and Genetically Modified CD34 Bone Marrow Cells, Following Gamma Irradiation in Cynomolgus Macaques - Genetic Engineering: Recent Developments in Applications - page 139

replication in simian cells is located at or near the cyclophilin A (CyPA) bind-
ing region of the capside protein [45]. The hydrophobic pocket of cyclophilin A
(CypA) makes direct contact with an exposed, proline-rich loop on HIV-1 capsid
(CA) and renders reverse transcription complexes resistant to an antiviral activity
in human cells. A CypA fusion with TRIM5 (a member of the tripartite motif
family) that is unique to New World owl monkeys also targets HIV-1 CA, but this
interaction potently inhibits infection. A similar block to HIV-1 infection in Old
World monkeys is attributable to the α isoform of the TRIM5 orthologue in these
species and using RNA interference techniques, Berthoux et., al demonstrated
that CypA inhibits HIV-1 replication in these cells because it is required for CA
recognition by TRIM5 α [46]. SIV vectors can also efficiently transduce human
cells[33,47], and may therefore prove a useful alternative to HIV-1-based vectors,
at least in the early phase of preclinical testing of lentivirus vectors. We found
that the proportion of eGFP-positive cells obtained before myeloid differentia-
tion (mean value of 30%) was similar to that obtained with CD34+ cells from
human donors transduced with lentiviral [48-51], retroviral [52-54], AAV[55],
or adenovirus/AAV-derived [56] vectors. However, it is possible to increase the
transduction rate, such that 90% transduced human CD34+ cells are obtained
from cord blood, 80% from bone marrow and 75% from G-CSF mobilized pe-
ripheral blood [57]. We analyzed transduction in two types of assay, based on
committed (CFC) and primitive (LTC-IC) hematopoietic progenitors, as analyses
of the transduction of committed progenitors only bear little relation to the trans-
duction efficiency for stem cells and less differentiated cells in the long term. Af-
ter myeloid differentiation, eGFP+ cells were detected, in similar proportions, in
CFC on day 15 and LTC-IC on day 50 after transduction, indicating that the vec-
tor was able to transduce progenitor cells and most immature hematopoietic cells
with a similar efficiency. Similar results have been reported for stimulated human
CFC and LTC-IC, which were found to be transduced with similar efficiency by a
lentiviral vector based on HIV-1[58]. In this previous study, significant resistance
to lentiviral transduction was reported in unstimulated primitive human cells.
These results may explain why, in our study, the use of cytokines during trans-
duction made possible the genetic modification of LTC-IC, which are quiescent.
Cytokine treatment may have led to these cells entering the cell cycle, facilitating
transduction. This result confirms the greater efficiency of lentiviral vectors than
of retroviral vectors for the transduction of CD34+ cells. Nevertheless, in our
study, only half as many eGFP-positive cells were obtained after differentiation
as were obtained from undifferentiated CD34+ cells. Similar observations have
been made with MLV-transduced progenitor cells from human donors[59]. We
demonstrate here that these differences may be accounted for by the pseudotrans-
duction detected at 24 h of incubation with the vector, confirming the results
reported with CD34+ cells in studies using VSVg-pseudotyped MLV-derived[60]