In VitroAssembly 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 beta-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.

The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells.

“…Forstova et al (1995) demonstrated that murine polyomavirus VP1 pseudocapsids generated from a recombinant baculovirus are able to transfer heterologous DNA efficiently into mammalian cells. More recently, SV40 pseudovirions self-assembled in insect cells have also been shown to be able to introduce foreign genes into mammalian cells (Sandalon et al, 1997). The current study demonstrates that human JCV VP1 is able to form pseudovirion and pseudocapsid particles when expressed in E. coli.…”

“…The size of DNA molecules packaged in viral particles after self-assembly in insect cells seems to vary. Sandalon et al (1997) showed that the capsid-like particles EE Self-assembly of JCV VP1 in E. coli Self-assembly of JCV VP1 in E. coli formed by SV40 VP1 expressed in insect cells can package various lengths of DNA in vitro, although the viral genome is about 5 kbp. One possible explanation is that eukaryotic DNA may associate with host histone proteins to form a minichromosome conformation.…”

SV40 Pseudovirion gene delivery of a toxin to treat human adenocarcinomas in mice

“…Dithiothreitol, PMSF and the protease inhibitor cocktail were added to the buffer immediately before use. 14,15 Nuclear extract concentration was measured using the BCA Protein Assay protocol (Pierce, Rockford, IL). The nuclear extract was kept for more than a year at À201C.…”

Efficient gene transfer using the human JC virus-like particle that inhibits human colon adenocarcinoma growth in a nude mouse model

“…Since then, progress has been made in the use of polyomavirus capsid as a gene delivery vector. [18][19][20][21][22][23][24][25][26][27] However, the efficiency of gene transduction using polyomavirus vector is low. In part, this may have been caused by inefficient DNA packaging.…”