Enhancement by polylysine of transient, but not stable, expression of genes carried into cells by polyoma VP1 pseudocapsids

Abstract

Gene transfer to provide long-term expression of a therapolylysine, unencapsidated DNA was shown to be propeutic product, without introducing unwelcome genetic tected against DNase activity, but electron microscopy information, is a goal being sought for therapy of both her-(EM) revealed the formation of large mixed aggregates. editary and acquired diseases. Polyoma virus pseudocap-The addition of pseudocapsids to these aggregates, and sids, generated from a VP1-expressing recombinant bacumeasurement of mobilities of the complexes in CsCl equililovirus, lack viral DNA and have been successfully used to brum centrifugation, indicated that they contained negliintroduce small exogenous genes stably into cells in vitro gible amounts of VP1. For subsequent pseudofection by a process designated 'pseudofection'; although pseudoexperiments, DNA was complexed first with pseudocapcapsids protect only about 3 kbp of exogenous DNA, low sids, then polylysine was added. The latter did not appear efficiency transfer of a larger fragment (6.2 kbp) has been to displace pseudocapsids from DNA, and was found to observed. Here, expression of a 7.2 kbp plasmid (pCMV␤) increase the efficiency of short-term expression both in encoding the ␤-galactosidase gene was assessed to moniin vitro and in vivo experiments. Gene expression, anator not only efficiency, but the ability of pseudocapsids to lysed histochemically or by the polymerase chain reaction, transfer larger-sized DNA on their own, or in the presence revealed transcriptional activity of the input gene, with of the polycation, poly-L-lysine, added to protect nonexpression first diminishing, then stabilising over time. The encapsidated DNA. When complexed to pseudocapsids presence of pseudocapsids, in complexes with DNA with only, the efficiency of expression of the transferred ␤-or without polylysine, allowed for stable and persistent galactosidase gene (in human or rodent cells), although gene expression. low, appeared to stabilise with time. In the presence of

Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: Application to single virus-like particle entry into a cell

“…Polyoma VLPs are nanospheres composed of 360 molecules of a single viral coat protein, VP1, that have been shown to interact with and carry plasmid DNA into cells (12,13). The protein nanospheres encapsidate 1-2 kb of the plasmid DNA whereas the remainder loosely associates with the outside of the VLP structure (14,15). These particles enter cells by both receptor-dependent and -independent routes, but only the former is productive for gene transfer (16).…”

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.…”

Sustained ex vivo and in vivo transfer of a reporter gene using polyoma virus pseudocapsids

“…20,21 Expression of the ␤ galactosidase gene transferred by pseudocapsids to monkey epithelial cos7 cells, in transient assays, appears to occur at a low level, being identified in a few cells only per culture when detected with the chromogenic substrate for ␤ galactosidase, X-gal 22 ( Figure 1, panel Gene Therapy out in a system where the input plasmid was amplified, more cells were found to be positive. For example, pseudocapsid-mediated DNA transfer to monkey cos7 cells (constitutively expressing the SV40 large T antigen 23 ) with a plasmid encoding the SV40 origin of replication and the gene for green fluorescent protein (pEGFP), resulted in detection of the expressed gene in at least 0.1-0.5% of the cells after 24 h, whereas calcium phosphate transfection resulted in 1-5% of positively staining cells (panels c and d, respectively).…”

“…Pseudocapsid/DNA complexes produced by this method have been shown to mediate gene transfer in several tissue culture systems, including cell lines of human origin, 13 and transfer can be further augmented by creating ternary complexes with poly-l-lysine. 20 Since pseudocapsids can deliver DNA to cells in culture, the question was raised whether transfer could also be achieved in more complex systems, for example, in tissues in vivo. The ␤ galactosidase gene was selected as a marker for these experiments, allowing for both histochemical and PCR-based assays for measuring DNA transfer.…”

“…13,20 This method is based on virus-like particles, composed solely of VP1. They are efficiently generated in insect cells infected with a recombinant baculovirus encoding the VP1 gene under the control of a strong promoter.…”