We are developing a retroviral replicating vector (RRV) encoding
cytosine deaminase as an
anticancer agent for
gliomas. Despite its demonstrated natural selectivity for
tumors, and other safety features, such a virus could potentially cause off-target effects by productively infecting healthy tissues. Here, we investigated whether incorporation of a hematopoietic lineage-specific
microRNA target sequence in RRV further restricts replication in hematopoietic lineage-derived human cells in vitro and in murine lymphoid tissues in vivo. One or four copies of a sequence perfectly complementary to the guide strand of
microRNA 142-3p were inserted into the
3' untranslated region of the RRV genome expressing the transgene encoding
green fluorescent protein (GFP). Viral spread and GFP expression of these vectors in hematopoietic lineage cells in vitro and in vivo were measured by qPCR, qRT-PCR, and flow cytometry. In hematopoietic lineage-derived human cell lines and primary human stimulated peripheral blood mononuclear cells, vectors carrying the 142-3pT sequence showed a remarkable decrease in GFP expression relative to the parental vector, and viral spread was not observed over time. In a syngeneic subcutaneous mouse
tumor model, RRVs with and without the 142-3pT sequences spread equally well in
tumor cells; were strongly repressed in blood, bone marrow, and spleen; and generated
antiviral immune responses. In an immune-deficient mouse model, RRVs with 142-3pT sequences were strongly repressed in blood, bone marrow, and spleen compared with unmodified RRV. Tissue-specific
microRNA-based selective attenuation of RRV replication can maintain
antiviral immunity, and if needed, provide an additional safeguard to this delivery platform for gene therapy applications.