Optimization of retroviral gene transfer protocol to maintain the lymphoid potential of progenitor cells.
We have attempted to improve retrovirus-mediated gene transfer efficacy into hematopoietic progenitor cells (HPCs) without causing them to lose their lymphoid potential. Highly purified CD34(+) cells on CH-296 fibronectin fragments have been transduced with three different cytokine combinations. Murine CD2 was used as a marker gene. Transgene expression was assayed by FACS analysis shortly after transduction of CD34(+) cells and after long-term culture (LTC) extended by differentiation of various lymphoid lineages: NK cells, B cells, and dendritic cells. Compared with the historical cytokine mix, i.e., SCF (stem cell factor) + IL-3 (interleukin 3) + IL-6, the combination SCF + FL (Flt-3 ligand) + M-GDF (megakaryocyte growth and differentiation factor) + IL-3 significantly improved the total number of viable cells and CD34(+) cells after transduction and the long term-cultured progenitors after 6 weeks. In addition, the combination of SCF + FL + M-GDF + IL-3 maintained more efficiently the lymphoid potential of the progeny of transduced long term-cultured CD34(+) cells, as attested by the significantly higher number of CD56(+), CD19(+), and CD1a(+) cells recovered when FL and M-GDF were added to SCF + IL-3. Thus, even though additional improvements may still be needed in transduction of HPCs, these conditions were adopted for a clinical trial of gene therapy for X-linked severe combined immunodeficiency.
Hacein-Bey
S
S
Unité Inserm U429, Laboratoire de Thérapie Génique, Hôpital Necker, 75743 Paris Cedex 15, France. sbey@wanadoo.fr
Gross
F
F
Nusbaum
P
P
Hue
C
C
Hamel
Y
Y
Fischer
A
A
Cavazzana-Calvo
M
M
D000818:Animals; D018949:Antigens, CD1 / Q000378:metabolism; D018941:Antigens, CD19 / Q000378:metabolism; D018952:Antigens, CD34 / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism; D018801:CD2 Antigens / Q000378:metabolism; D019002:CD56 Antigen / Q000378:metabolism; D002469:Cell Separation; D016207:Cytokines / Q000378:metabolism; D003713:Dendritic Cells / Q000378:metabolism; D005312:Fetal Blood / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D007377:Interleukin-3 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007694:Killer Cells, Natural / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology* / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D010641:Phenotype; D012190:Retroviridae / Q000235:genetics*; D019089:Stem Cell Factor / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology; D013926:Thrombopoietin / Q000378:metabolism; D013997:Time Factors; D014161:Transduction, Genetic; D019076:Transgenes
D018949:Antigens, CD1; D018941:Antigens, CD19; D018952:Antigens, CD34; C419393:CD1a antigen; D018801:CD2 Antigens; D019002:CD56 Antigen; D016207:Cytokines; D005353:Fibronectins; D007377:Interleukin-3; D015850:Interleukin-6; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D013926:Thrombopoietin