In Vitro Assembly of SV40 Virions and Pseudovirions: Vector Development for Gene Therapy

Abstract

SV40 is an attractive potential vector with high-efficiency gene transfer into a wide variety of human tissues, including the bone marrow, a critical target organ for the cure of many diseases. In the present study, the three SV40 capsid proteins, VP1, VP2, and VP3, were produced in Spodoptera frugiperda (Sf9) insect cells. Their co-production led to spontaneous assembly of SV40-like particles. Nuclear extracts containing the three proteins were allowed to interact with purified SV40 DNA, or with plasmid DNA produced and purified from Escherichia coli. The 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, supercoiled DNA is being packaged and transmitted into the target cells. The transmitted DNA is biologically functional in gene expression and replication. The process, which utilizes naked DNA, is not dependent on the SV40 packaging signal ses. The procedure allows packaging of plasmids significantly larger than SV40 and permits the inclusion of potent regulatory signals, such as β-globin locus control region (LCR) elements. These studies are the first step in the development of purified, in vitro-constructed pseudovirions for experimental and medical use.

Overview summary

SV40 pseudovirions are potentially suitable vectors for somatic gene therapy due to their efficient gene delivery into a wide spectrum of cell types. The ideal, safe way to prepare pseudovirions for human treatment is by in vitro packaging, because all steps of the preparation can be well controlled. Here we demonstrate that SV40-like pseudovirions can be produced from purified DNA and cell-free extracts containing recombinant viral capsid proteins. The particles are infectious and are capable of transmitting functional genes into target cells. These studies are the first step in the development of purified, in vitro-constructed pseudovirions for experimental and for medical use.