title: High-efficiency transduction of primary human hematopoietic stem cells and erythroid lineage-restricted expression by optimized AAV6 serotype vectors in vitro and in a murine xenograft model in vivo.
abstract: We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34(+) cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34(+) cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease.
authors.affiliation: Experimental Hematology Laboratory, Department of Physiology, School of Basic Medical Sciences, Central South University, Changsha, China.
authors.forename: Liujiang
authors.initials: L
authors.lastname: Song
authors.forename: Xiaomiao
authors.initials: X
authors.lastname: Li
authors.forename: Giridhara R
authors.initials: GR
authors.lastname: Jayandharan
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.forename: George V
authors.initials: GV
authors.lastname: Aslanidi
authors.forename: Chen
authors.initials: C
authors.lastname: Ling
authors.forename: Li
authors.initials: L
authors.lastname: Zhong
authors.forename: Guangping
authors.initials: G
authors.lastname: Gao
authors.forename: Mervin C
authors.initials: MC
authors.lastname: Yoder
authors.forename: Changquan
authors.initials: C
authors.lastname: Ling
authors.forename: Mengqun
authors.initials: M
authors.lastname: Tan
authors.forename: Arun
authors.initials: A
authors.lastname: Srivastava
mesh_terms: D000818:Animals; D019070:Cell Lineage; D000229:Dependovirus / Q000145:classification / Q000235:genetics*; D041905:Erythroid Cells / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000821:virology; D006801:Humans; D020014:K562 Cells; D051379:Mice; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D012703:Serotyping; D014158:Transcription, Genetic; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes / Q000235:genetics; D014183:Transplantation, Heterologous