Soluble SV40 capsid proteins were obtained by expression of the three late genes, VP1, VP2, and VP3, in Sf9 cells using baculovirus expression vectors. Coproduction of the capsid proteins VP1, VP2, and VP3 was achieved by infecting Sf9 cells with the three recombinant baculovirus species at equal multiplicities. All three proteins were found to be localized in the nuclear fraction. Electron microscopy of nuclear extracts of the infected cells showed an abundance of SV40-like capsid structures and heterogeneous aggregates of variable size, mostly 20-45 nm. Under the same staining conditions wild-type SV40 virions are 45 nm. The capsid-like particles sedimented in glycerol gradients similarly to authentic wild-type SV40 virions. Pentamers of the major capsid protein VP1 were also seen. Protein analysis on sucrose gradients demonstrated that the capsid-like particles can be disrupted by treatment with the reducing agent dithiothreitol and the calcium chelator EGTA. The capsid-like particles were found to be significantly less stable than SV40 virions and were partially stabilized by calcium ions. Understanding the complex interactions between the capsid proteins is important for the development of an efficient in vitro packaging system for SV40 virions and pseudovirions.

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.

In vitro packaging of plasmid DNA using recombinant SV40 capsid proteins is a potentially useful procedure that overcomes some restrictions of the other SV40 systems such as the requirement for SV40 sequences and the limitation in size of DNA that can be packaged. The in vitro packaging system uses the four SV40 proteins (VP1, VP2, VP3, and agno) or VP1 only. The ability to confer drug resistance by three ABC transporter genes (MDR 1, MRP 1, or MXR) was determined using the surrogate fluorescent substrates rhodamine-123 or calcein AM and their specific inhibitors, or by using specific antibodies to the transporters to detect cell surface expression by fluorescence-activated cell sorter analysis (FACS). A green fluorescent protein plasmid (EGFP-C1) was also used to monitor gene transfer. The packaged plasmids ranged in size from 4.2 to 17.6 kb, and only slightly affected particle size as determined by electron microscopy. When 9.5 kb and larger plasmids were packaged using all SV40 proteins, MDR1 expression was decreased compared to VP1 alone. The size of the 15.2 kb DNA after packaging was the same as the original DNA. Packaging with SV40 capsid proteins in vitro does not require any SV40 sequences. Using either the MDR1 or the GFP gene we could demonstrate enhanced expression when cells were pretreated with phorbol 12-myristate 13-acetate (PMA) at low concentrations. Interferon-gamma did not alter expression. We conclude that in vitro packaging is more flexible then previously realized, permitting packaging of at least 17 kb plasmid DNA without the requirement for any viral sequences. This system combines efficient gene delivery of the SV40 viral vector with the presumed safety of nonviral vectors.

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We previously reported that administration of an adeno-associated virus 2 (AAV2) vector encoding a rat tumor necrosis factor (TNF) receptor-immunoglobulin Fc (TNFR:Fc) fusion gene to rats with streptococcal cell wall-induced arthritis resulted in suppression of joint inflammation and cartilage and bone destruction, as well as expression of joint proinflammatory cytokines. In this study, we used an alternate rat model of arthritis to compare the serum levels and duration of TNFR:Fc protein expression following intramuscular administration of pseudotyped AAV-TNFR:Fc vectors based on serotypes 1, 2, and 5. All three pseudotyped AAV-TNFR:Fc vectors led to sustained expression of serum TNFR:Fc protein for at least one year. Serum TNFR:Fc protein levels in rats administered intramuscularly with AAV2/1-TNFR:Fc vector were up to 100- and 10-fold higher than in rats administered the AAV2-TNFR:Fc or AAV2/5-TNFR:Fc vectors, respectively. A single intramuscular administration of AAV2/1-TNFR:Fc vector at vector doses ranging from 10(10) to 10(12) DNase-resistant particles (DRP) per animal, resulted in complete and long-term suppression of recurrent joint inflammation for at least 150 days. Our results establish a proof of concept for administration of an AAV2/1-TNFR:Fc vector to the muscle to achieve long-term, sustained and therapeutically relevant levels of TNFR:Fc protein to treat chronic systemic inflammatory joint diseases.