Design of Ad5F35 vectors for coordinated dual gene expression in candidate human hematopoietic stem cells

Objective

Adenoviral vector−mediated gene expression is an attractive approach to manipulate or report gene expression in human hematopoietic stem cells (HSC) when transient gene expression is preferred. Previous studies have demonstrated that fiber-retargeted Ad5F35 vectors can mediate efficient gene transfer into human HSCs. In this study, we investigated the potential of bi-directional promoter-controlled Ad5F35 vector for coordinated dual gene expression in candidate HSCs.

Materials and Methods

We have engineered Ad5F35-ΔLNGFR-BiDp encoding kinase domain deleted low-affinity NGF receptor (ΔLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter, which is composed of human phosphoglycerate kinase promoter and minimal core promoter from human cytomegalovirus. The expression pattern of ΔLNGFR and GFP following Ad5F35-ΔLNGFR-BiDp gene transfer in various cell types, including candidate HSCs, was compared to Ad5F35-ΔLNGFR-IRES vector encoding phosphoglycerate kinase promoter-controlled bicistronic expression cassette for ΔLNGFR and GFP.

Results

Using Ad5F35-ΔLNGFR-BiDp, we demonstrated a coordinated, high-level dual gene expression in leukemic cells and cord blood CD34⁺ cells. However, the ability of Ad5F35-ΔLNGFR-BiDp-GFP for coordinated dual gene expression varied significantly between repopulating progenitor cells. In nonobese diabetic severe combined immune deficient mice bone marrow transplantation assay, sorted CD34⁺/ΔLNGFR⁺/GFP⁺ cells following infection with Ad5F35-ΔLNGFR-BiDp showed predominantly myeloid lineage reconstitution with limited lymphoid lineage differentiation capacity, whereas the CD34⁺/ΔLNGFR⁺/GFP⁻ cells exhibited both myeloid and lymphoid reconstitution.

Conclusions

This study indicates that bi-directional promoter-controlled Ad5F35 vector, such as Ad5F35-ΔLNGFR-BiDp, can be particularly useful for manipulation of myeloid progenitor cells and potentially in myeloid lineage leukemic cells as well.