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progenitors that were already committed and fewer pluripotent stem cells ca-
pable of long-term reconstitution than medullary HSC[69]. The small numbers
of eGFP-producing cells observed in our study may be due to an anti-eGFP im-
mune response. Some reports have suggested that such reactions do not generally
occur after irradiation[70], but two reports described the induction of cytotoxic
T-lymphocyte responses to enhanced green (GFP) or yellow (YFP) fluorescent
proteins after myeloablative conditioning. One of these reports concerned ba-
boons that had received primitive hematopoietic cells transduced with HIV-1-
based lentiviral vectors[71] and the other concerned rhesus macaques that had
received CD34+ stem cells transduced with a retroviral vector[72].
Lentiviruses, like retroviruses, can be used to integrate transgenes into the
host genome. Two severe adverse events occurred in two patients in the SCID-X1
gene therapy trial 30 to 34 months after injection of the autologous CD34+ cells
corrected using a retroviral vector. In these patients, an uncontrolled clonal T
lymphoproliferative syndrome, similar to acute lymphoblastic leukemia, was ob-
served [73,74]. This study highlights the risk of insertional mutagenesis restricted
to retroviral and lentiviral gene transfer. In the future, additional safety measures
could be considered, such as the use of self-inactivating LTRs (as in our study) to
reduce enhancer activity, the addition of insulators to reduce the risk further, and
the insertion of a second transgene encoding a “suicide” product, such as herpes
thymidine kinase, making it possible to kill the transduced cells with ganciclovir.
Unlike studies in mice, in which the follow-up period is necessarily limited, stud-
ies in large animals, with a longer life span, are compatible with more extensive
follow-up. The development of linear amplification-mediated PCR (LAM-PCR),
a sensitive and robust approach to molecular clonal analysis, has made it pos-
sible to identify and analyze the contribution of individual transduced clones to
hematopoiesis. Clonal analysis may provide information about the dominance of
transduced clones, potentially predicting possible progression or the propensity
to develop clonal hematopoiesis and leukemia. Moreover, replication-competent
retrovirus (RCRs), recombinant retrovirus and interaction with endogenous ret-
roviruses should also be investigated, when evaluating the biosafety of retrovirus
and lentivirus.
conclusion
The results reported here provide the first evidence that gene transfer into medul-
lary hematopoietic stem cells and long-term expression of the transgene are pos-
sible, using an SIV-based lentiviral vector in non human primates, which provide
the best clinical models for in vivo evaluation of the feasibility and safety of gene
therapy strategies.
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