IN-VITRO ASSEMBLY OF SV40 VIRIONS AND PSEUDOVIRIONS - VECTOR DEVELOPMENT FOR GENE-THERAPY

SV40 is an attractive potential vector with high-efficiency gene trans fer into a wide variety of human tissues, including the bone marrow, a critical target organ for the cure of many diseases. In the present s tudy, the three SV40 capsid proteins, VPI, VP2, and VP3, were produced in Spodoptera frugiperda (Sf9) insect cells. Their co-production led to spontaneous assembly of SV40-like particles. Nuclear extracts conta ining the three proteins were allowed to interact with purified SV40 D NA, or with plasmid DNA produced and purified from Escherichia coli. T he experiments demonstrated a physical association between the DNA and capsid proteins, protection from DNase I digestion, and the formation of infectious particles. The results indicate that intact, supercoile d DNA is being packaged and transmitted into the target cells. The tra nsmitted DNA is biologically functional in gene expression and replica tion. The process, which utilizes naked DNA, is not dependent on the S V40 packaging signal ses. The procedure allows packaging of plasmids s ignificantly larger than SV40 and permits the inclusion of potent regu latory signals, such as beta-globin locus control region (LCR) element s. These studies are the first step in the development of purified, in vitro-constructed pseudovirions for experimental and medical use.