Design and construction of oncoretroviral vectorsexpressing a packageable ribonuclease for use inHIV gene therapy

摘要

A number of different strategies are being developed for inhibition of human immunodeficiency virus type-1 (HIV- 1) replication via gene therapy. In this study, a packageable ribonuclease, Gag-RNase T1, was constructed. The Gag domain from HIV-1 should allow copackaging into HIV-1 virions and the RNase T1 domain from Aspergillus oryzae should allow cleavage of HIV-1 virion RNA. In order to have regulator of expression of virion proteins (Rev)- dependent and Rev-independent production of Gag-RNase T1, the HIV-1 Rev responsive element (RRE) and the mason-pfizer monkey virus (MPMV) constitutive transport element (CTE) were cloned downstream to the gagt1 gene. Expression and enzymatic activity of the Gag-RNase T1 fusion protein was compared using the Moloney murine leukemia virus (MoMuLV)-based vector, MoTiN, and murine stem cell virus (MSCV)-based vector, MGIN. Very little amount of Gag-RNase T1 was present in the cell lysate and in the culture supernatant of cells co- transfected with the MoTiN-based vector. In contrast, the amount of Gag-RNase T1 present in the cell lysate and in the culture supernatant of cells co-transfected with the MGIN-based vector was ~20 fold better. HIV-based lentiviral vector particles produced from cells expressing Gag-RNase T1 or mutant Gag-RNase T1 were also analyzed. Gag-RNase T1 present in these samples was shown to be full-length (56 kDa) and was enzymatically active in vitro. However, the titer of these vector particles was not decreased. These results suggest that Gag-RNase T1 is only capable of homochimeric assembly and is excluded from vector particles containing the HIV-1 Gag and Gag-Pol proteins.