High Cloning Capacity ofIn VitroPackaged SV40 Vectors with No SV40 Virus Sequences

Abstract

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.

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

“…Moreover, it does not require any SV40 sequence, thus providing efficient gene delivery at the same level of safety when using nonviral vectors. [11][12][13] In this study, we demonstrate delivery of PE38 toxin with or without a combination of the chemotherapeutic agent doxorubicin. SV40 pseudovirion delivery of PE was found to be effective in the treatment of human adenocarcinomas growing in mice, either by direct injection or systemically.…”

Virus-like particles production in green plants

“…These VLPs are composed of 90 dimers of the virus coat protein arranged in a T=3 symmetry; they are stable at low pH and to lyophilization, and easily produced and purified in large quantities using the baculovirus expression system [14]. The VLP vaccine stimulated systemic and mucosal immune response when administered orally in mice [8] and humoral, mucosal and cellular immune response in human volunteers [10,15].…”

“…Particularly attractive is their use in transporting viral vectors for gene therapy and DNA vaccines, exploiting in some cases the natural tropism of the viral particles. It has been reported that supercoiled plasmid DNA up to 17 kb was encapsidated in vitro inside simian virus 40 capsid proteins [15] and that the HPV-16 L1 VLPs are able to package unrelated plasmid DNA in vitro and then to deliver this foreign DNA to eukaryotic cells with the consequent expression of the encoded gene. The gene transfer rate observed was higher than DNA delivered alone or with liposome [16].…”

SV40 reporter viruses

“…SV40 ST is also encoded by this construct. Although there do not appear to be major size constraints to DNA packaging into SV40 particles in vitro (Kimchi-Sarfaty et al, 2003), an effort was made to keep the GFP recombinant close to the size of WT SV40. The genome of SV40vGFP is only 90 nucleotides smaller than the WT viral DNA.…”