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.
Zhong
Li
L
Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Li
Weiming
W
Li
Yanjun
Y
Zhao
Weihong
W
Wu
Jianqing
J
Li
Baozheng
B
Maina
Njeri
N
Bischof
Daniela
D
Qing
Keyun
K
Weigel-Kelley
Kirsten A
KA
Zolotukhin
Irene
I
Warrington
Kenneth H
KH
Li
Xiaomiao
X
Slayton
William B
WB
Yoder
Mervin C
MC
Srivastava
Arun
A
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