In vivo selection of hematopoietic stem cells transduced at a low multiplicity-of-infection with a foamy viral MGMT(P140K) vector.
OBJECTIVE
Using a clinically relevant transduction strategy, we investigated to what extent hematopoietic stem cells in lineage-negative bone marrow (Lin(neg) BM) could be genetically modified with an foamy virus (FV) vector that expresses the DNA repair protein, O(6)-methylguanine DNA methyltransferase (MGMT(P140K)) and selected in vivo with submyeloablative or myeloablative alkylator therapy.


METHODS
Lin(neg) BM was transduced at a low multiplicity-of-infection with the FV vector, MD9-P140K, which coexpresses MGMT(P140K) and the enhanced green fluorescent protein, transplanted into C57BL/6 mice, and mice treated with submyeloablative or myeloablative alkylator therapy. The BM was analyzed for the presence of in vivo selected, MD9-P140K-transduced cells at 6 months post-transplantation and subsequently transplanted into secondary recipient animals.


RESULTS
Following submyeloablative therapy, 55% of the mice expressed MGMT(P140K) in the BM. Proviral integration was observed in approximately 50% of committed BM-derived progenitors and analysis of proviral insertion sites indicated up to two integrations per transduced progenitor colony. Transduced BM cells selected with submyeloablative therapy reconstituted secondary recipient mice for up to 6 months post-transplantation. In contrast, after delivery of myeloablative therapy to primary recipient mice, only 25% survived. Hematopoietic stem cells were transduced because BM cells from the surviving animals reconstituted secondary recipients with MGMT(P140K)-positive cells for 5 to 6 months.


CONCLUSIONS
In vivo selection of MD9-P140K-transduced BM cells was more efficient following submyeloablative than myeloablative therapy. These data indicate that a critical number of transduced stem cells must be present to produce sufficient numbers of genetically modified progeny to protect against acute toxicity associated with myeloablative therapy.
Cai
Shanbao
S
Department of Pediatrics, James Whitcomb Riley Hospital for Children and Indiana University School of Medicine, Indianapolis, IN 46202-5525, USA.
Ernstberger
Aaron
A
Wang
Haiyan
H
Bailey
Barbara J
BJ
Hartwell
Jennifer R
JR
Sinn
Anthony L
AL
Eckermann
Olaf
O
Linka
Yvonne
Y
Goebel
W Scott
WS
Hanenberg
Helmut
H
Pollok
Karen E
KE
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology* / Q000821:virology*; D019070:Cell Lineage / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012192:Retroviridae Infections / Q000821:virology; D053150:Simian foamy virus / Q000201:enzymology* / Q000235:genetics
D019853:O(6)-Methylguanine-DNA Methyltransferase