Systematic determination of the packaging limit of lentiviral vectors

Because of their ability to transduce nondividing cells, human immunodefici ency virus type 1 (HIV)-based vectors have great potential for the therapeu tic delivery of genes to cells. We describe here a systematic study of the packaging limit of HIV-based vectors. Restriction endonuclease-generated ba cterial chromosomal DNA fragments of different lengths were cloned at three different positions within a lentiviral vector. Vesicular stomatitis virus G protein (VSV G) pseudotyped lentiviral particles were prepared and the d ifferent clones were titered on mammalian cells. We observed that the restr iction endonuclease site positions at the 5' and 3' ends of the genome were superior with regard to insertional capacity of foreign DNA. In all cases, viral titers decreased semi-logarithmically with increasing vector length. There appears to be no absolute packaging limit because measurable titers were obtained even when the proviral length was in excess of 18 kb. The red uction in titer appears to occur at the level of viral encapsidation, altho ugh we cannot exclude limitations in nuclear export of proviral RNA. These results suggest that HIV-based vectors may have a secondary advantage over oncoretroviral vectors because of their greater packaging limit, although t he very low titers of the larger vectors will be of limited utility.