Adeno-associated virus 2-mediated transduction and erythroid lineage-restricted expression from parvovirus B19p6 promoter in primary human hematopoietic progenitor cells.
Human parvovirus B19 gene expression from the viral p6 promoter (B19p6) is restricted to primary human hematopoietic cells undergoing erythroid differentiation. We have demonstrated that expression from this promoter does not occur in established human erythroid cell lines in the context of a recombinant parvovirus genome (Ponnazhagan et al. J Virol 69:8096-8101, 1995). However, abundant expression from this promoter can be readily detected in primary human bone marrow cells (Wang et al. Proc Natl Acad Sci USA 92:12416-12420, 1995; Ponnazhagan et al. J Gen Virol 77:1111-1122, 1996). In the present studies, we investigated the pattern of expression from the B19p6 promoter in primary human bone marrow-derived CD34+ HPC undergoing differentiation into myeloid and erythroid lineages. CD34+ cells were transduced with recombinant adeno-associated virus 2 (AAV) vectors containing the beta-galactosidase (lacZ) gene under the control of the following promoters/enhancers: the cytomegalovirus promoter (vCMVp-lacZ), B19p6 promoter (vB19p6-lacZ), B19p6 promoter with an upstream erythroid cell-specific enhancer element (HS-2) from the locus control region (LCR) from the human beta-globin gene cluster (vHS2-B19p6-lacZ), and the human beta-globin gene promoter with the HS-2 enhancer (vHS2-beta p-lacZ). Transgene expression was evaluated either 48 h after infection or following erythroid differentiation in vitro for 3 weeks. Whereas high-level expression from the CMV promoter 48 h after infection diminished with time, low-level expression from the B19p6 and the beta-globin promoters increased significantly following erythroid differentiation. Furthermore, in HPC assays, there was no significant difference in the level of expression from the CMV promoter in myeloid or erythroid cell-derived colonies. Expression from the B19p6 and the beta-globin promoters, on the other hand, was restricted to erythroid cell colonies. These data further corroborate that the B19p6 promoter is erythroid cell-specific and suggest that the recombinant AAV-B19 hybrid vectors may prove useful in gene therapy of human hemoglobinopathies in general and sickle cell anemia and beta-thalassemia in particular.
Kurpad
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C
Department of Microbiology & Immunology, Walther Oncology Center, Indiana University School of Medicine, Indianapolis 46202-5120, USA.
Mukherjee
P
P
Wang
X S
XS
Ponnazhagan
S
S
Li
L
L
Yoder
M C
MC
Srivastava
A
A
D000755:Anemia, Sickle Cell / Q000235:genetics / Q000628:therapy; D018952:Antigens, CD34 / Q000032:analysis; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D003587:Cytomegalovirus / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D015672:Erythroid Precursor Cells / Q000378:metabolism / Q000821:virology; D004920:Erythropoiesis / Q000235:genetics*; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D005814:Genes, Viral; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics*; D005914:Globins / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000821:virology; D006801:Humans; D007763:Lac Operon; D009928:Organ Specificity; D016732:Parvovirus B19, Human / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics; D017086:beta-Thalassemia / Q000235:genetics / Q000628:therapy
D018952:Antigens, CD34; D011993:Recombinant Fusion Proteins; D005914:Globins; D001616:beta-Galactosidase