Efficient and durable gene marking of hematopoietic progenitor cells in nonhuman primates after nonablative conditioning.
Optimization of mobilization, harvest, and transduction of hematopoietic stem cells is critical to successful stem cell gene therapy. We evaluated the utility of a novel protocol involving Flt3-ligand (Flt3-L) and granulocyte colony-stimulating factor (G-CSF) mobilization of peripheral blood stem cells and retrovirus transduction using hematopoietic growth factors to introduce a reporter gene, murine CD24 (mCD24), into hematopoietic stem cells in nonhuman primates. Rhesus macaques were treated with Flt3-L (200 microgram/kg) and G-CSF (20 microgram/kg) for 7 days and autologous CD34(+) peripheral blood stem cells harvested by leukapheresis. CD34(+) cells were transduced with an MFGS-based retrovirus vector encoding mCD24 using 4 daily transductions with centrifugations in the presence of Flt3-L (100 ng/mL), human stem cell factor (50 ng/mL), and PIXY321 (50 ng/mL) in serum-free medium. An important and novel feature of this study is that enhanced in vivo engraftment of transduced stem cells was achieved by conditioning the animals with a low-morbidity regimen of sublethal irradiation (320 to 400 cGy) on the day of transplantation. Engraftment was monitored sequentially in the bone marrow and blood using both multiparameter flow cytometry and semi-quantitative DNA polymerase chain reaction (PCR). Our data show successful and persistent engraftment of transduced primitive progenitors capable of giving rise to marked cells of multiple hematopoietic lineages, including granulocytes, monocytes, and B and T lymphocytes. At 4 to 6 weeks posttransplantation, 47% +/- 32% (n = 4) of granulocytes expressed mCD24 antigen at the cell surface. Peak in vivo levels of genetically modified peripheral blood lymphocytes approached 35% +/- 22% (n = 4) as assessed both by flow cytometry and PCR 6 to 10 weeks posttransplantation. In addition, naïve (CD45RA(+) and CD62L(+)) CD4(+) and CD8(+) cells were the predominant phenotype of the marked CD3(+) T cells detected at early time points. A high level of marking persisted at between 10% and 15% of peripheral blood leukocytes for 4 months and at lower levels past 6 months in some animals. A cytotoxic T-lymphocyte response against mCD24 was detected in only 1 animal. This degree of persistent long-lived, high-level gene marking of multiple hematopoietic lineages, including naïve T cells, using a nonablative marrow conditioning regimen represents an important step toward the ultimate goal of high-level permanent transduced gene expression in stem cells.
Rosenzweig
M
M
New England Regional Primate Research Center, Harvard Medical School, Southborough, MA, USA.
MacVittie
T J
TJ
Harper
D
D
Hempel
D
D
Glickman
R L
RL
Johnson
R P
RP
Farese
A M
AM
Whiting-Theobald
N
N
Linton
G F
GF
Yamasaki
G
G
Jordan
C T
CT
Malech
H L
HL
D000818:Animals; D015703:Antigens, CD / Q000032:analysis / Q000235:genetics*; D001402:B-Lymphocytes / Q000166:cytology / Q000502:physiology; D001854:Bone Marrow Cells / Q000166:cytology; D051927:CD24 Antigen; D005434:Flow Cytometry / Q000379:methods; D005819:Genetic Markers; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006098:Granulocytes / Q000166:cytology / Q000502:physiology; D006410:Hematopoiesis / Q000187:drug effects; D019650:Hematopoietic Stem Cell Mobilization; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007937:Leukapheresis / Q000379:methods; D008253:Macaca mulatta; D008297:Male; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000494:pharmacology; D009000:Monocytes / Q000166:cytology / Q000502:physiology; D016133:Polymerase Chain Reaction; D012190:Retroviridae; D019089:Stem Cell Factor / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology / Q000502:physiology; D020858:Tissue and Organ Harvesting / Q000379:methods; D014916:Whole-Body Irradiation
D015703:Antigens, CD; D051927:CD24 Antigen; C497889:CD24 protein, human; D005819:Genetic Markers; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D016179:Granulocyte Colony-Stimulating Factor