Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 241

the cells for relatively short periods of time, and then discarding the cells and thaw-
ing a new sample [165] . Vectors produced from mouse cells were most sensitive
regardless of the virus used, whereas vectors produced from particular human cells
were less sensitive. Amphotropic MLV was less resistant to human complement than
the endogenous cat virus RD114.
Sensitivity to complement is due to direct lysis of virions and to indirect lysis
involving anti- O -galactosyl antibodies prevalent in human serum [166] . Use of packag-
ing cells derived using human cells and RD114 virus (FLYRD18 cells; 166) improves
gene transfer in human trials. Packaging cells based on the 10A1 virus are also avail-
able for gene transfer into cells where the relative levels of Ram-1 and Glvr-1 are not
known [167] . The packaging cell line GP7C-tTA-G10 is also designed to express the
VSV-G protein that increases the host range of the vector and, because of stability
imparted by VSV-G, makes it possible to concentrate viral stocks. Replication-
competent retrovirus (RCR) has been produced by recombination between the short
homologous regions of nucleotide sequences in the retroviral vector and packaging
cell line. The removal of overlapping sequences between the vector and the packaging
constructs is crucial for minimizing the possibility of homologous recombination, and
therefore, the production of RCR [168] . MLV-based retroviral vectors are widely used
gene delivery vehicles for gene therapy clinical trials, being engaged in almost 70% of
approved protocols. The viral components of these vectors comprise transcomplement-
ing genes ( gag , pol , and env ) expressed in packaging cells and the cis -acting sequences
(LTRs, primer binding sites, and packaging signals) present within the vector. Inclusion
of part of the gag gene sequence in vectors was shown to increase recombinant viral
titer. This gag sequence was reported to increase the amount of vector packaged into
virions and was therefore thought to be an extension of the packaging signal. However,
retention of the gag sequence in vectors is not desirable as it could aid recombination
in the packaging cells, leading to generation of replication-competent viruses. There
are many problems with the retroviral vectors currently in clinical use, such as MFG
and LN-based vectors [167,169] .
5.4.2.3 Vector Production Techniques
Two methods have been used to introduce vectors into packaging cells [168] :
1. Direct transfection of DNA into the cells: Insertion of multiple copies of the vector into
the recipient cells may lead to the production of both unrearranged and rearranged viruses.
This method leads to higher viral titers because of the presence of multiple copies of the
vector.
2. Infection with a virus produced by DNA transfection of other packaging cells: Insertion of
only single integrated proviruses, which can be screened for the proper structure, generally
yields an unrearranged virus.
Once a packaging cell line has been isolated that produces helper-free unrearranged
vector at high titer, this line may provide a continuous source of the vector, but some
instability of viral production by packaging cell lines has been observed. Prolonged
passage of packaging cells and vector-producing packaging cells should be avoided
because this practice also minimizes the possibility of helper virus production due to