Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5.
Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.
Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Li
L
Zhong
Weiming
W
Li
Yanjun
Y
Li
Weihong
W
Zhao
Jianqing
J
Wu
Baozheng
B
Li
Njeri
N
Maina
Daniela
D
Bischof
Keyun
K
Qing
Kirsten A
KA
Weigel-Kelley
Irene
I
Zolotukhin
Kenneth H
KH
Warrington
Xiaomiao
X
Li
William B
WB
Slayton
Mervin C
MC
Yoder
Arun
A
Srivastava
D000818:Animals; D000067696:Ataxin-1; D000067528:Ataxins; D002478:Cells, Cultured; D004274:DNA, Recombinant / Q000008:administration & dosage; D000229:Dependovirus / Q000145:classification / Q000235:genetics* / Q000276:immunology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000821:virology; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D009713:Nucleotidyltransferases / Q000235:genetics; D054578:Protein Tyrosine Phosphatase, Non-Receptor Type 2; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D013234:Stem Cells / Q000378:metabolism* / Q000821:virology; D014161:Transduction, Genetic; D019076:Transgenes / Q000502:physiology
D000067696:Ataxin-1; D000067528:Ataxins; C000593750:Atxn1 protein, mouse; D004274:DNA, Recombinant; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D019009:Proto-Oncogene Proteins c-kit; D009713:Nucleotidyltransferases; C057535:lincosaminide O-nucleotidyltransferase; D054578:Protein Tyrosine Phosphatase, Non-Receptor Type 2; D017027:Protein Tyrosine Phosphatases; C516653:Ptpn2 protein, mouse