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 126

We report here the results obtained in vitro and in vivo in an experiment as-
sessing the efficacy and safety of a gene transfer protocol based on the transduc-
tion of simian CD34+ bone marrow cells with a minimal SIVmac251-derived
lentiviral vector. This system is based on the VSVg-pseudotyped SIV vector en-
coding enhanced green fluorescent protein (eGFP) under control of the phospho-
glycerate kinase (PGK) promoter. Most immature CD34+ hematopoietic cells
capable of initiating long-term culture (LTC-IC) were efficiently transduced, and
eGFP-positive cells were detectable in vivo in all animals more than one year after
transplantation.
Methods
Animals
Male cynomolgus macaques (Macaca fascicularis), weighing between 3 and 6 kg
were imported from Mauritius and housed in single cages within level 3 bio-
safety facilities, according to national institutional guidelines (Commission de
génie génétique, Paris, France). All experimental procedures were carried out in
accordance with European guidelines for primate experiments (Journal Officiel
des Communautés Européennes, L358, December 18, 1986).
Immunoselection of Non Human Primate CD34 + bone
Marrow Progenitor cells
Bone marrow mononuclear cells were obtained from the iliac crest or by as-
piration from the humerus and isolated by standard Ficoll density-gradient
centrifugation (MSL2000, Eurobio, Les Ulis, France). Cells were washed
twice in phosphate-buffered saline (PBS, Eurobio, Les Ulis, France) and re-
suspended in 1% FCS (Fetal Calf Serum; Bio West, France) in PBS. The
cellular fraction was then enriched in CD34 + cells by positive immuno-
magnetic selection, using beads coupled to a specific antibody (clone 561;
Dynabeads M-450 CD34, Progenitor Cell Selection System, Dynal, Oslo,
Norway), according to the manufacturer' s instructions. Immunoselected
CD34 + cells were stained with a specific PE-conjugated anti-CD34 antibody
(clone 563; Pharmingen, Becton Dickinson, California, USA) and analyzed
by flow cytometry (LSR, Becton Dickinson, California, USA) to evaluate the
level of enrichment. All preparations contained more than95% CD34 + cells,
with a mean value of 97% ± 1% (n = 12) for in vitro assays and 96% ± 1%
(n = 4) for in vivo assay.