Factors influencing the titer and infectivity of lentiviral vectors.

Lentiviral vectors have undergone several generations of design improvement to enhance their biosafety and expression characteristics, and have been approved for use in human clinical studies. Most preclinical studies with these vectors have employed easily assayed marker genes for the purpose of determining vector titers and transduction efficiencies. Naturally, the adaptation of these vector systems to clinical use will increasingly involve the transfer of genes whose products may not be easily measured, meaning that the determination of vector titer will be more complicated.

Integrated self-inactivating lentiviral vectors produce full-length genomic transcripts competent for encapsidation and integration.

To make human immunodeficiency virus type 1 (HIV-1)-based vectors safer for use in the research and clinical setting, a significant modification to the HIV-1 genome has been the deletion of promoter and enhancer elements from the U3 region of the long terminal repeat (LTR). Vectors containing this deletion are thought to have no LTR-directed transcription and are called self-inactivating (SIN) lentivectors. Using four distinct approaches, we show that SIN lentivectors continue to have promoter activity near the 5' LTR, which is responsible for the production of full-length vector transcripts.

The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells.

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a.